ABSTRACT
Data on the morphological development of inbred and F1 hybrid mice reared at 22·8 °C dry bulb, 17·8 °C wet bulb, and 32·2 °C dry bulb, 26·7 °C wet bulb are presented. Animals were weighed at birth, 1 and 2 weeks of age and at each developmental stage. All animals were examined for the freeing of the ear pinnae, eruption of the lower incisor teeth and perforation of the eye membranes and in addition females were examined for the appearance of the first pair of nipples and for perforation of the outer part of the vagina.
Within each environment, but especially at 23 °C, there are genotypically characteristic differences in the ages at maturation of each variable. Though the ages of maturation of the different variables are highly correlated within genotypes, early maturation of one variable is not necessarily associated with the early maturation of others. Reciprocal F1 hybrid differences indicate a maternal effect upon the age of maturation which is more marked at 23 than at 32 °C.
Comparisons of the ages at maturation in the two environments showed that the only character to mature earlier at 23 than at 32 °C was pinna freeing. The most marked influence of the 32 °C environment in advancing development was on the appearance of nipples and on vaginal opening. In almost all cases, animals reared at 32 °C were lighter at the time of maturation than those at 23 °C. Hence when allowance was made for environmental differences in body weight the association between an environment at 32 °C and early development was further enhanced. Those animals that were relatively heavy when weighed at the weekly interval prior to maturation matured earlier than the lighter ones, especially at 23 1C, indicating that morphological maturation was closely related to overall growth.
Both the within, and between, litter variation in the ages and body weights at maturation of a strain tended to be greater in animals exposed to the higher temperature. In bilaterally distributed characters, asymmetrical maturation was more common at 32 than at 23 °C.
INTRODUCTION
The effects of environmental and genetic variation on mammalian growth and size have been investigated (Mills, 1945; Cawley, McKeown & Record, 1954; Acheson, 1960; Hammond, 1961; Harrison, 1963; Chevillard, Portet & Cadot, 1963; Porter & Festing, 1969) but little attention has been paid to their effects on postnatal morphological development. These are of interest for, in principle, they afford a means of analysing the extent to which the correlations between maturation patterns and growth can be broken and the extent to which the processes can be shown to be independent.
Where morphological maturation has been investigated in relation to the body weights of rats or mice, results varied according to the conditions in which the animals were maintained and to the particular morphological characteristics observed (Engle & Rosasco, 1927; Parkes, 1929; Outhouse & Mendel, 1933; Kennedy, 1957; Biggers, Ashoub, McLaren & Michie, 1958; Widdowson & McCance, 1960; Knudsen, 1962; Barnett & Burn, 1967; Gall & Kyle, 1968). Among mice the issue is complicated by the existence of differences in the ages at morphological maturation among inbred strains (van Ebbenhorst-Tengbergen, 1942; Yoon, 1955) and between reciprocal F1 hybrids (Deringer, Heston & Andervont, 1945). These genetic observations have not been related to differences in the growth patterns of the various genotypes.
This paper is concerned with examining, among inbred strains of mice and the F1 hybrids between them, the effects of a difference in environmental temperature upon the time of appearance of a number of external morphological characters, and relating these measures of development to variation in the overall growth of the animals, as assessed from body weight.
MATERIALS AND METHODS
The general design of the study and the environmental specifications have been detailed elsewhere (Garrard, Harrison & Weiner, 1974a). In summary, animals were bred and reared either at 22·8 ± 1·6 °C dry bulb, 17·8 ± 1·0 °C wet bulb or at 32·2 ± 0·5 °C dry bulb and 26·7 ± 0·5 °C wet bulb. At 23 °C the following inbred strains were observed: C57BL, BALB/c and CBA/H. The latter strain included, mainly because of its availability, an isogenic substrain with a T6 translocation, CBA/H.T6T6 (referred to as CBA/T6). The six reciprocal F1 hybrids derived from these inbred strains (but using only CBA/T6 to represent the CBA genotype) were also reared and are identified as IF1 = C57 × CBA/T6, IIF1 = C57 × BALB and IIIF1= CBA/T6 × BALB. At 32 °C observations were limited to C57, BALB and reciprocal crosses of the hybrid between them, i.e. IIE1. Data on C57 are limited because of the difficulty experienced in breeding and rearing this strain at high temperatures (Garrard et al. 1974a).
The developmental features observed were the chronological ages at which freeing of the ear pinnae, eruption of the lower incisor teeth, and the first perforation of the eye membranes occurred. These were recorded for males and females but two additional characters were scored for females: the age of appearance of the first pair of nipples (usually one of the inguinal pairs) and the age at perforation of the vagina. The latter is not the same stage as that identified by other authors, which more closely coincides with the first oestrus cycle, but is the time when the external part of the vagina is perforated. This, like the other morphological features examined, matures in the preweaning period.
Animals were examined each morning to determine any changes in their developmental status which had occurred over the preceding 24 h. Since there was not always symmetry in the development of two of the bilateral characters, pinna freeing and eye opening, separate observations were made for the right and left sides. When development was asymmetrical the mean age at maturation for each animal was used in the statistical analysis.
At each morphological developmental stage the body weight of an individual was recorded to 0·5 g, a mean value again being calculated when development was asymmetrical. Where the character was observed in both sexes the data for males and females have been combined as analysis showed no statistically significant sex effect. Animals were also weighed at birth, and at 1 and 2 weeks of age.
RESULTS
Morphological maturation at 23 °C
Data on the ages at morphological maturation of male and female litter mates have been combined to give means since no sex differences were detected. These means have been used to derive the mean ages at which the ear pinnae became free, the lower incisor teeth erupted and the eye membranes became perforated in offspring of each of the mating types (see Table 1). Mean strain values for the ages of appearance of nipples and the opening of the external part of the vagina, in females, are also included in the table. Genotypic comparisons of the mean ages at maturation have been made and statistically significant differences are set out in Table 2. The morphological character whose developmental age was most influenced by genotype was the eruption of the lower incisor teeth, while the age at pinna freeing was least responsive to genotype.
Significant differences in the mean ages at maturation of offspring of different mating types reared at 23 °C

BALB is particularly advanced, as compared with all other genotypes in the eruption of the lower incisors. With the exception of opening of the eyes, the development of BALB litters generally tends to be advanced with respect to that of the other inbred strains, especially CBA and CBA/T6. Some consistent hybrid genotypic differences which emerged were the relatively early development of IF1; (C57 × CBA/T6) litters and the late development of IIIF1 (CBA/T6 ×BALB) litters. Among litters with a CBA/T6 mother there was a marked tendency for inbred offspring to mature earlier than hybrid offspring.
Inter-litter variation in the ages at maturation of five of the characters tends to be low but is greater at the time of vaginal opening than at development of the remaining four characters. Certain strain differences in inter-litter variation in ages at maturation were found, and can be summarized as follows. Both the inbred and hybrid litters of BALB mothers usually mature at a more uniform age than litters of C57 mothers. BALB litters are also less variable than CBA/T6 litters, but in this case a reciprocal hybrid CBA/T6 × BALB difference did not emerge.
Inter-character correlations between developmental ages at 23 °C
The relationship between the ages of individual animals, within a mating type, at maturation of pairs of morphological characters have been estimated by calculating correlation coefficients. The coefficients are presented in Table 3. Data for males and females are combined. Between most of these pairs of characters there is a highly significant correlation but especially between the times of pinna freeing and eye opening. For some characters the offspring of certain mating types may show high correlations, while in others the correlations are not statistically significant. Some of the non-significant correlations may be attributable to the very small variance in the time of maturation of some of the characters.
The effects of body weight on maturation at 23 °C
One factor which might have produced the observed correlations between the ages at maturation of the various morphological characters is the integrated nature of general growth. The relationship between body weight and age at maturation was examined by linear regression analysis using the mean weights of litters at that weekly age which immediately preceded the development of the character and the mean ages of litters at the time of maturation. For pinna freeing and eye opening there is a statistically significant, negative regression coefficient relating age of maturation and body weights at fixed ages in all mating types. The majority of the remaining negative regressions are also significant for the other three characters. On the basis of this analysis it is evident that, within genotypes, light animals matured later than heavy ones.
To some extent the relationship between weight growth and maturation explains the observed differences in the mean ages at maturation of the different mating types. Few of the regression coefficients vary significantly according to mating type, but the regression constants do vary with mating type. For instance, among reciprocal cross hybrids, none of the regression coefficients differ significantly from one another but there are significant maternal effects upon the intercepts for each parameter.
Ages at morphological maturation have also been related to the body weights of animals at the time of maturation. The mean weights at the times of maturation are included in Table 1. Although the relationship between weight and age at maturation is less than between weight at a fixed age and age at maturation, some of the coefficients for the former are nevertheless significant for some characters in some mating types. This is particularly evident at eye opening when, with only one exception, there is a highly significant negative regression between the age of development of the character, and body weight at that age. Pinna freeing shows the same phenomenon, though to a smaller extent. In contrast, appearance of nipples, eruption of lower incisors and, to some extent, opening of the vagina are more affected by weight growth, there being very few significant regression coefficients between ages at development of these characters and body weights at the time of maturation.
Maturation at 32 °C
The mean ages of litters reared at 32 °C at the time of maturation of five morphological characteristics are shown in Table 4. There was much less variation between the offspring of the different mating types at 32 than at 23 °C. Ages at pinna freeing, nipple appearance and vaginal opening did not vary with mating type, although ages at lower incisor eruption and eye opening did so. Incisor eruption occurred later in C57 than in BALB (P = < 1·0%) and in the reciprocal cross hybrids maturation occurred at the same age, which was intermediate between that of the two parental strains. In contrast, at eye opening there was a reciprocal cross difference (P =< 5·0%). For this character both the inbred and F1 hybrid litters of C57 mothers matured before the inbred and hybrid litters of BALB mothers.
There are few systematic differences in within, and between, litter variances that can be related to mating type, although between litter variances in the times of nipple appearance and lower incisor eruption were significantly higher in BALB than in the reciprocal cross hybrids. BALB also had high levels of within litter variation in the age of development of the same two characters, as compared with the hybrid offspring of BALB mothers.
Inter-character correlations between developmental ages at 32 °C
The correlation coefficients relating the ages at maturation of pairs of characters are presented in Table 5. With the exception of C57, for which the data are too limited, in those characteristics which are common to both males and females the coefficients are all positive and almost all are highly significant. The relationship between ages at nipple appearance and vaginal opening is not significant in hybrids. Contrary to the results obtained at 23 °C and to other results from animals reared at 32 °C, there is a significant (P = < 5·0%) inverse relationship between ages at pinna freeing and nipple appearance among each of the reciprocal cross hybrids.
Correlations between the ages at development of five parameters of maturation, for individuals reared at 32 °C

At 32 °C many of the coefficients for the linear regressions of age at maturation on body weight at the immediately preceding weekly interval do not differ significantly from zero. Where coefficients are significant they are negative, indicating that lighter animals matured later than heavy ones. With the exception of the difference between the inbred and hybrid litters of BALB mothers at vaginal opening, the relationship between the weights of animals at a fixed age and their ages at maturation of a particular morphological character is not influenced by genotype.
Variation in age at maturation, with respect to body weight at that age, was also examined by linear regression analysis. There was no evidence at 32 °C for the negative weight effect that was found at 23 °C. Indeed the only significant regression coefficient occurred in BALB, at opening of the vagina, and the relationship was positive.
Comparison of maturation at 23 and 32 °C
Among the offspring of the four mating types which were reared at both temperatures, the mean ages at freeing of the ear pinnae were always significantly less at 23 °C than in animals reared at 32 °C. However, the appearance of nipples and opening of the vagina occurred earlier at 32 °C (P = < 1·0%, in each comparison). A similar retardation in development at 23 °C was apparent at eye opening, though only in BALB and hybrid litters of C57 mothers was the difference statistically significant (P = < 5·0% and <0·1% respectively). The environmental effect upon the age of eruption of the lower incisors was less marked, the only significant difference occurring in C57 mothers’ hybrid offspring, with animals at 32 °C maturing the earlier.
In almost all cases animals reared at 23 °C were heavier, at the time of maturation, than those reared at 32 °C. Mean body weights at the times of nipple appearance and vaginal opening particularly evidenced the environmental effect for animals not only matured at an earlier age at 32 °C but they also matured at a lighter body weight. Regression analysis showed that lower incisor eruption occurred earlier in animals at 32 °C than in animals of the same weight and genotype reared at 23 °C. Further, the effect of the higher temperature in advancing eye opening becomes even more marked when environmentally associated differences in body weight are taken into account. Consideration of body weight does not modify the conclusion that pinna freeing occurred earlier at 23 than at 32 °C.
Some genotypic differences between the ages at maturation of particular characters were altered by the environmental temperature. For example, for all the morphological characters except eye opening BALB animals reared at 23 °C tended to mature earlier than the other inbred lines but this was not so at 32 °C, though the data for C57 are scant. Other genotypic relationships in ages at maturation were unaffected by the temperature of the environment. Whilst maternal and genotypic offspring effects were detected among hybrids, in both environments there were no consistent differences in the ages at maturation of inbred and hybrid lines.
Age at maturation was also regressed on litter size at birth and, as was to be expected from the relationship between body weight and litter size at birth (Garrard, Harrison & Weiner 1974b), there was a marked effect of litter size on developmental age. Although these regressions did not distinguish hybrids from inbreds any more clearly than did the weight regressions, there was evidence that at 23 °C, though not at 32 °C animals from inbred lines mature later than F1 hybrids from litters of the same size.
Comparisons of the between, and within, litter variances of both the ages and the body weights at which maturation occurred showed that the environment affected the magnitude of the variance. Where significant differences occurred it was usually the variance of animals reared at 32 °C which was the greater. This was true of between litter variances at pinna freeing, lower incisor eruption and nipple appearance among the offspring of BALB mothers. The hybrid offspring of C57 mothers were exceptional, their between litter variation in age at nipple appearance being significantly greater at 23 than at 32 °C. Estimates of within litter variation in age at maturation were greater at 32 °C at pinna freeing and eye opening for BALB and reciprocal IIF1(C57/BALB) hybrids but the environmental effect was reversed at nipple appearance and vagina opening.
The environmental effect upon the symmetry of maturation
Two of the characters that were considered in the previous analyses, pinna freeing and eye opening, have a bilateral disposition which leant itself to scoring for symmetry or asymmetry in development. When the development was asymmetrical the interval between development on the two sides rarely exceeded one day. A contingency analysis was used to assess the effects of genetic and environmental variation on the numbers of symmetrically and asymmetrically developing animals in the two environments. Within neither environment did the frequency of asymmetrical maturation vary according to mating type but there were significant differences according to the temperature at which the animals had been reared. The freeing of the pinnae and eye opening were less symmetrical in their development among BALB animals reared at 32 °C than when reared at 23 °C (P = <5·0% and 0·5%, respectively). Among the reciprocal IIF1 (C57/BALB) hybrids, those with a C57 mother showed a greater asymmetry at pinna freeing (P = < 0·5%) and those with a BALB mother at eye opening (P = <0·5%) at 32 than at 23 °C. There is thus evidence that asymmetrical maturation is more common in the hotter conditions.
DISCUSSION
This study has demonstrated that the age of maturation of certain external morphological features is influenced by genetic and environmental factors. These factors may interact with each other so that the relative ages at maturation of mice of different strains varies according to the environmental temperature. Other investigations have shown that environmental conditions, especially in association with nutritional variation (Parkes, 1929; Yoon, 1955; Kennedy, 1957; Widdowson & McCance, 1960), affect the age of maturation of some of the characters that were also examined in this study. Age at maturation was shown to depend upon growth in body weight. In the present study too the ages at eruption of the lower incisor teeth, the appearance of the nipples and, to a lesser extent, the opening of the vagina are closely related to general growth in animals reared at 23 °C. In contrast, ages at pinna freeing and eye opening of the same animals indicate that the development of these characters tends to be at a fixed age, the age being strain-specific. Nevertheless, for all the morphological characters examined there is evidence that slowly growing animals, those with low body weights at the fixed weekly intervals, mature morphologically more slowly than rapidly growing animals.
From studies in which morphological development was examined in relation to environmental temperature variation earlier eye opening (Biggers et al. 1958) and vaginal opening (Biggers et al. 1958; Knudsen, 1962) in hot than in temperate conditions were associated with comparatively high body weights. However, the temperate conditions which these workers employed were less stressful than those in the present study, and promoted weight growth. In the work reported here high temperatures caused early maturation in these characters and also in nipple appearance and lower incisor eruption, even when early postnatal growth in weight was reduced. Certainly at the time of maturation, animals reared at 32 °C weighed less than those reared at 23 °C. The time at maturation of four of the characters which developed earlier at 32 than at 23 °C coincided with that period when the growth rate was greater in the animals reared at 32 °C (Garrard et al. 1974b). The one character that matured earlier at 23 °C – pinna freeing – did so in the first week when the 32 °C environment had a retarding effect on growth, as assessed from body weight. Despite this and the high correlations between weights and ages at maturation within each environment, the pattern of growth in body weight cannot fully account for the observed environmental and genotypic differences in maturation. Under the stress of an environmental temperature of 32 °C there is evidence for a breakdown in the relationship between the pattern of growth in body weight and the maturation of particular morphological characters that was found at 23 °C. Just what is responsible for the dissociation of weight growth and morphological maturation is obscure. There is some evidence that specific growth factors may be involved in the maturation of some of the tissues. Cohen (1962), for instance, has been able to isolate from the submaxillary gland of the adult male mouse a factor which, when injected into neonates, causes premature incisor eruption and eye opening. It is possible that environmental temperature could affect the production of, or receptivity of tissue to, such factors. Since a cold environment retards development independently of its effect on body weight (Biggers et al. 1958; Barnett & Coleman, 1959) there is evidence for a specific temperature effect, with the heat promoting and the cold retarding maturation.
Even more marked than the environmental temperature effects are the differences in the developmental ages of offspring of the different mating types, especially at 23 °C. A few differences appear to be related to maternal environmental factors acting mainly, though not invariably, on weight growth. In addition, there is evidence for offspring genotypic effects, not only among the different inbred strains, but also between F1 hybrids of different genotypes. The latter are most manifest at pinna freeing and eye opening, when a C57 contribution to a hybrid, irrespective of whether it is derived from a maternal or a paternal source, advances maturation; whilst a BALB contribution retards development. These patterns are not predictable from the relative developmental ages of the respective inbred parental strains although these strains are themselves variable. Thus BALB, which is characterized by the early maturation of most parameters, is markedly retarded in eye opening. When allowance is made for the relationship between body weight growth and maturation age, the development of BALB is not as precocious as would at first appear. There are, however, problems in assessing the effects of body weight, for the analysis has shown that although weight growth and maturation within any one strain are closely related, the form of the relationship is a characteristic of the genotype.
There is very little evidence from either environment for systematic differences in the rates of maturation of hybrids as a whole as compared with all the inbred strains. The type of variation occurring within and between the inbreds and hybrids reared at 23 °C is much more consistent with specific hétérozygotie/homozygotic contrasts but before this can be firmly concluded a detailed genetic analysis is required. Specific genetic effects might be expected to operate either through variable receptivity of tissues to local morphogenetic factors or through quantitative variation in the production of such factors. At a developmental level the genetic variation could therefore be causally produced in much the same way as the environmental effect. However, the data from animals reared at 32 °C, though much more limited, indicate that genetic variety is less expressed in age at maturation in this environment than at 23 °C. So far as can be judged the maturation promoting effect of the hotter environment occurs more or less independently of genotype.
The results indicate that, while growth and the features of morphological development that have been examined are closely interrelated, at least some of the underlying processes are different and can be independently affected by genetic and environmental factors. Although the growth pattern has been described in terms of weights at particular ages or when particular events occurred, the data indicate that even fine monitoring of differences in growth rates and changes in these rates would be unlikely to account for some of the observed independence between growth and maturation.
ACKNOWLEDGEMENTS
We are grateful to Mr M. Potter for his technical assistance and to the Science Research Council for a grant to G. Garrard.