1. The sera of rats at various stages of development from the 17th day of gestation to adulthood were examined by paper electrophoresis.

  2. A component distinct from, and of higher mobility than, the bulk of the albumin was found at the 17th and 18th days of development. However, the mobility of this component is well within the limits of mobility of adult albumin.

  3. Evidence supporting the existence of a component in the beta-globulin region specific to the perinatal animal is presented.

  4. Gamma globulin and an asymmetry associated with it were demonstrable in all stages examined. The possible relation of this asymmetry to eta globulin is discussed.

  5. Problems concerning the physiology of the foetus arising from the changes in the relative concentrations of the serum proteins are pointed out.

The morphological changes which occur during late embryonic and early postnatal life in common laboratory animals are, with one set of exceptions, now well understood. The exception relates to those changes which require the use of physiological, biophysical, or biochemical techniques. Outstanding among these are the changes in the relative concentrations of the serum proteins (Kekwick, 1959). Only for the chicken (Moore, Shen, & Alexander, 1945; Marshall & Deutsch, 1950; Heim & Schechtman, 1954; Weller & Schechtman, 1957) has a reasonably complete picture been built up.

In the case of the changes in the relative concentrations of the serum proteins of the late embryonic and early postnatal rat, the various investigations, including the more extensive ones of Jameson, Alvarez-Tostado, & Lew (1948), Shmerling & Uspenskaya (1955), and Gurvich & Karsaevskaya (1956), leave a confusing and contradictory picture due to the use of widely divergent methods and the limited number of stages investigated.

In view of this, a systematic survey of the serum proteins of the rat, from the earliest suitable developmental stage to the immediate postnatal stages, was undertaken with the aid of a highly standardized technique. Sera of adult male and female rats were also examined as controls and for purposes of comparison.

All animals used were Sprague-Dawley rats, obtained from the Holtzman Company, or their descendants, bred in this laboratory. The animals were between 90 and 200 days old when mated. They were maintained on an ad libitum diet of Purina rat chow and water supplemented with occasional feedings of raw pork-liver, hard-boiled egg-slices, or slices of orange. The date of insemination was determined by the finding of spermatozoa in daily vaginal smears. Pregnancy was assumed to have begun at about 7 a.m. on the morning on which spermatozoa were found in the smear. In order to maintain proper timing, all bleeding was done between 7 and 8 a.m. Delivery was assumed to have occurred, and generally did occur, early during the 23rd day of pregnancy. This day is designated hereafter as the first day post-partum.

Blood was obtained in one of three ways. In the procedure used with most foetuses, the mother was anesthesized with ether, her abdominal and uterine cavities opened, and the foetuses successively removed from their membranes. A small, canoe-shaped piece of lucite was slipped under the umbilical cord. After drying off the outside of the cord, one or more vessels within it were nicked and the blood collected in a siliconed pipette as it escaped into the plastic boat. In the cases of some of the older foetuses and of the new-born rats, a deep dorsal incision was made into the neck, care being taken to avoid cutting into the trachea or oesophagus. The first drop of blood to appear was usually discarded and the next few drops collected directly into a siliconed centrifuge tube. The blood from at least four foetuses or neonatal animals from the same litter was pooled. The immature and adult animals were bled by direct heart-puncture under light ether anesthesia. In all cases, the blood was allowed to clot for approximately 4 hours in siliconed centrifuge tubes at about 4° C. After brief centrifugation, the supernatant serum was removed and its refractive index determined with white light in an Abbe-type refractometer. No dilutions were made.

Paper electrophoresis was carried out in accordance with the instructions (Publication RIM-5, 1957) for, and utilizing the equipment of, the Spinco model R system. The buffer was barbital (5,5-di-ethylbarbituric acid) 2·76 g.; barbital sodium (2-sodium, 5,5-di-ethylbarbituric acid) 15-40 g.; distilled water to make one litre. This buffer is 0·075 M in the sodium salt and has a pH of 8-6. 0-010 ml. of adult serum was applied to each paper strip. The volume of serum from the earlier stages applied to each strip was adjusted so that the amount of protein, as estimated from the refractive index, was approximately equal to that in 0·010 ml. of adult protein. This precaution proved necessary because the resultant pattern is somewhat dependent on the amount of protein applied. A potential gradient of 2·6 v./cm., resulting in a current flow of 0·31 ma. per strip, was applied under constant current conditions for 19 hours. After staining with bromphenol blue, photometric evaluation was performed with the Spinco Analytrol. Components were delimited by dropping verticals (Tiselius & Kabat, 1939) and the relative concentrations were calculated from the ratio of the area under each section of the curve to that under the total curve.

Typical patterns obtained from the sera of the various developmental stages examined are shown in Text-fig. 1. The leading component visible in the patterns of animals of 17 and 18 days of gestation has an anodic mobility well within the range shown by adult albumin. An asymmetry on the trailing edge of the gamma globulin component is evident at all stages. The leading edge of the beta globulin fraction shows considerable asymmetry in the neonatal and immature stages. No significant changes in the mobilities of the components were detected.

Text-fig. 1.

Typical patterns of sera obtained at various stages of development. Component 1: fast albumin. Component 2: albumin. Component 3: alpha-1 globulin. Component 4: alpha-2 globulin. Component 5: alpha-3 globulin. Component 6: beta globulin. Component 7: gamma globulin.

Text-fig. 1.

Typical patterns of sera obtained at various stages of development. Component 1: fast albumin. Component 2: albumin. Component 3: alpha-1 globulin. Component 4: alpha-2 globulin. Component 5: alpha-3 globulin. Component 6: beta globulin. Component 7: gamma globulin.

The percentage composition of the serum proteins at the various developmental stages examined is given in Table 1 and the changes in these percentages during development are shown in Text-fig. 2. Albumin constitutes the largest single component at all stages. The fast albumin fraction loses its identity as a component distinct from the main mass of albumin between the 18th and 19th days of gestation. The changes in the relative concentrations of albumin and alpha-1 globulin are generally opposite to each other. Gamma globulin does not begin to approach adult level until after at least one month of postnatal life.

Table 1.

Relative concentration of the serum protein fractions

Relative concentration of the serum protein fractions
Relative concentration of the serum protein fractions
Text-fig. 2.

Average percentage composition of electrophoretic components in rat serum from the 17th day of incubation to the mature adult. The short vertical lines indicate the range of plus or minus one standard deviation. The dashed lines connect points plotted on an interrupted time scale and hence their slope does not represent a rate of change.

Text-fig. 2.

Average percentage composition of electrophoretic components in rat serum from the 17th day of incubation to the mature adult. The short vertical lines indicate the range of plus or minus one standard deviation. The dashed lines connect points plotted on an interrupted time scale and hence their slope does not represent a rate of change.

Gurvich & Karsaevskaya (1956) report the presence of a pre-albumin component in the sera of rat embryos weighing from 0·43 to 0·72 g. According to the data of Stotsenburg (1915), such animals would be between about and days of gestation. The average weight for rats of 17 days of gestation was found to be 0·74±0·05 g. in the present experiment, thus confirming the above age estimates. It is in this same age-range, and only in this age-range, that the fast albumin component was detected in the present work. The relative concentration of the pre-albumin listed by Gurvich & Karsaevskaya (1956) ranges from 16·00 to 21 ·0 per cent., whereas the largest average value measured in the present work was 11 ·3±2-9 per cent. It is likely, therefore, that the pre-albumin fraction of these workers and the fast albumin component found in the present work include, at least in part, the same protein groups. The term ‘pre-albumin’ refers to a protein fraction having a greater anodic mobility at a pH above its isoelectric point than does the family of proteins designated as albumin. Such components are found, for example, in the sera of chicken embryos and laying hens (Heim & Schechtman, 1954). In the present case, however, the leading edge of the component in question does not exhibit a greater mobility than that of the leading edge of the albumin fraction of the adult or of later foetal stages. Secondly, it may be seen from Text-fig. 2 that the disappearance of the fast albumin on the 19th day of gestation is accompanied by a nearly proportional increase in the relative concentration of the albumin. This suggests that the material previously recognized as fast albumin does not disappear at all but that it becomes merged with the main bulk of the albumin group. If the fast albumin material had disappeared, all remaining components would be expected to show a compensatory increase in relative concentration, proportional to the fraction each constitutes of the total protein concentration. Actually, only two of the six components show any significant increase between the 18th and 19th days of development. Supporting evidence for the hypothesis that the fast albumin fraction becomes merged with the main body of albumin comes from the great variability in the relative concentration of albumin in the serum of the animals at the 19th day of gestation as shown by the high standard deviation value (Table 1). The loss of distinction between the fast albumin and the albumin may be due to the appearance of a relatively small amount of a new albumin sub-fraction having a mobility intermediate between those of the fast albumin and the peak of the main albumin mass. The appearance of new serum proteins during the course of rat ontogeny has been postulated on immunochemical grounds by Gurvich & Karsaevskaya (1956).

An asymmetry was found in the trailing region of the gamma-globulin fraction during all stages of development, including the adult. An asymmetry very similar in appearance and position was also found by Gurvich & Karsaevskaya (1956), but only in sera from perinatal animals. These workers have designated one component of this complex as eta globulin and consider it, on immunochemical grounds, to be a distinct protein family. Since in the present work, however, this component or asymmetry appears at all stages of development and always at or near the point of initial application of the serum, it is suggested that the asymmetry is an artifact due to adsorbed proteins. Gurvich & Karsaevskaya (1956) have recognized that, in their immunoelectrophoretic procedure, confusion can arise between an immunological precipitate and an adsorbed protein. Contrary to the findings of Shmerling & Uspenskaya (1955), gamma globulin could be demonstrated in the sera from all stages examined.

Shmerling & Uspenskaya (1955) report the presence, in embryos weighing 2 g. or more and in rats one day after birth, of an alpha-2 and an alpha-4 globulin which more or less completely replace the alpha-1 and alpha-3 globulins found in other developmental stages. They believe the former two components to be analogous to foetuin (Pedersen, 1947). Some support is lent to this view by the finding of a considerable degree of asymmetry in the beta-globulin peak of sera from perinatal and juvenile animals. The observation of these authors that the sera of suckling young are cloudy is confirmed, although occasionally nearly clear sera were obtained. Since optical clarity is not a prerequisite for paper electrophoresis, as it is for the classical Tiselius technique, no defatting process was used. The present work also confirms the view of these workers that, on the basis of their mobilities, the fractions of the same denomination in embryonic and adult sera are electrophoretically identical.

The observation that the relative concentration of gamma globulin of immature rats remains for an extended period below the levels found in the adult is in accord with the finding of Halliday & Kekwick (1957).

That the alpha-globulin fraction or series of fractions was found in the present work but not in that of Jameson, Alvarez-Tostado, & Lew (1948) may be due to the use of very different buffer systems.

The comparatively constant relative concentration of alpha-2, alpha-3, beta, and gamma globulins during intra-uterine existence may indicate that the embryonic pools of these components are in rather free communication with the corresponding, larger, maternal pools. Should this be the case, we would be presented with the physiologically interesting situation in which the internal environment of one organism, the foetus, changes in response to the demands placed upon, and the response pattern of, another organism, namely, the maternal one. The relations between the serum proteins of mother and foetus and the possible selective action of the placenta on the exchange of serum proteins is presently under study.

Les protéines du sérum du Rat pendant le développement

  1. Les sérums de rats ont été étudiés à différents stades du développement à partir du 17e jour de la gestation jusqu’à l’âge adulte par la méthode de l’électrophorèse sur papier.

  2. Un composant distinct et d’une plus grande mobilité que la plus grande partie de l’albumine à ce stade a été mis en évidence aux 17e et 18e jours du développement. Cependant, la mobilité de ce composant est bien dans les limites de mobilité de l’albumine adulte.

  3. Il y a de sérieuses raisons d’admettre l’existence d’un composant spécifique situé dans la région de la bêta-globuline, aux environs de la naissance.

  4. A tous les stades étudiés, on a démontré l’existence de gamma-globuline et de l’asymétrie qui lui est associée. La possibilité d’une relation de cette asymétrie avec l’eta globuline est discutée.

  5. L’accent est mis sur les problèmes concernant la physiologie du fœtus en rapport avec les changements dans les concentrations relatives des protéines du sérum.

This work was supported by research grants G-4821 and G-10104 from the National Science Foundation.

I gratefully acknowledge the technical assistance of Miss Frances Maleniak, M iss Carol Sperling, Miss Judith Krym, and Miss Janie Whittier.

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