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Pregnancy |
a Division of Molecular Biology and Genetics, Victorian Institute of Animal Science, Attwood, Victoria 3049, Australia
b Department of Zoology, The University of Melbourne, Victoria 3010, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, gene regulation, mammary glands
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Major changes to the amino acid pool in milk [11] can be explained by major changes in individual milk protein concentrations, many of which occur at the transition between the phases of lactation [2, 12]. At parturition, there is a coordinate induction of four major milk protein genes: 
-lactalbumin, ß-lactoglobulin, and - and ß-casein. Four to six days postpartum, the enzyme ß-1,3-galactosyltransferase is induced [13]. This enzyme appears to be related to induction of the sucking stimulus, the presence of which determines whether a gland develops or regresses [5, 14]. At the transition from phase 2A to phase 2B, when the young ceases to be permanently attached to the teat, expression of the early lactation protein (ELP) gene is down-regulated [15], and whey acidic protein (WAP) gene expression is induced [16]. At the transition from phase 2B to phase 3, as the young begin to exit the pouch, the WAP gene is down-regulated [16], late lactation protein (LLP)-A expression is up-regulated, and LLP-B is induced [2]. It is noteworthy that macropodid marsupials are able to undergo concurrent asynchronous lactation, whereby two young of different ages can suck from adjacent mammary glands that are secreting milk of different composition [7, 17, 18]. The correlation between putative changes in sucking pattern and changes in expression of individual milk protein genes during lactation suggests that the PY may play a critical role in regulating milk composition. In a number of species, sucking frequency is thought to control milk secretion by the effects of an autocrine factor, feedback inhibitor of lactation (FIL) [19], which has also been identified in tammar milk [20]. The FIL has an inhibitory effect on milk synthesis and secretion [21, 22] and has also been shown to alter mammary cell differentiation, metabolic activity, and possibly, prolactin receptor concentrations [23–25]. However, to our knowledge, it has not been established in the tammar whether this factor or, indeed, a similar mechanism regulates milk composition [20].
A preliminary study in which the sucking stimulus was maintained by fostering PY of reduced age onto lactating tammars suggested this species may regulate milk composition by a predetermined, inherent mechanism that begins at parturition [26, 27], thereby controlling the growth and development of the young by regulating both milk supply and milk composition [1, 6]. To test this hypothesis, groups of 60-day-old PY were fostered repeatedly into one group of host mothers to allow the lactational stage to progress 42 days ahead of the age of the young. The milk of both fostered and control groups was analyzed for changes in the secretory pattern of the major milk components.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tammar wallabies originating from Kangaroo Island (South Australia) were maintained in open grassy yards at Clayton (Victoria, Australia) as previously described [28]. Tammars at 60 days of lactation were divided into two groups of nine animals. All the PY of the host group were removed each fortnight over a period of 8 wk and replaced with a 60-day-old foster PY that had been removed from donor tammars (Fig. 1). The final fostered PY (at Day 116 of lactation) remained with the host mother for the remainder of the lactation period. The PY of the control group of tammars were also removed at fortnightly intervals but returned to their mothers. The host and control animal groups were maintained separately and milked at fortnightly intervals for the duration of the experiment. A third group of tammars was kept at the Victorian Institute of Animal Science (Attwood, Victoria, Australia), and individual tammars were milked once at the indicated stages of the lactation cycle. The reason for including the nonexperimental group was to confirm that no difference in milk composition existed during the lactation cycle between animals milked at fortnightly intervals and animals at comparable stages of lactation that had been milked only once.
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Tammars undergo a postpartum estrus, and the conceptus develops over the next 7–8 days into a new blastocyst that enters embryonic diapause [4]. To obtain accurately aged PY, pregnancies can be synchronized by removal of the PY. This reactivates the dormant blastocyst, resulting in parturition 26.5 days later [28, 29]. Thirty-six donor animals were synchronized to provide the four groups of 60-day-old PY for fostering. To confirm PY ages, head-length measurements were taken using digital calipers, and the ages were estimated from the growth curves of Poole et al. [30]. All animal experimentation was performed according to the procedures of the Victorian Institute of Animal Science, Animal Experimentation Ethics, and the University of Melbourne Animal Ethics Experimentation Committee.
Husbandry and Collection of Milk
The host and control tammars were milked at intervals of 2–3 wk from Days 60 to 289 of lactation (Fig. 1). At each milking event, the PY were removed from the teat by gentle pressure on the sides of the mouth, placed in labeled containers, and kept warm (32°C) and moist. As the PY advanced in age, grew fur, and became homeothermic (age, >180 days), they were held in calico sacks. After removal of the PY, the mothers were administered 0.2 IU of oxytocin (Heriot Agvet Pty. Ltd., Rowville, Australia) [18] and milked by gentle massage of the mammary gland and teat. Capillary tubes were used for collection of milk before Day 130 of lactation, and 1.5-ml microfuge tubes were used thereafter. Milk was stored at -20°C. The time during which the mother and young were separated was kept to a minimum, usually less than 30 min for PY younger than 130 days and 30–60 min for PY older than 130 days. This separation has no effect on growth of the young [31]. Mammary index (MI) measurements (i.e., maximum perpendicular dimensions of the mammary gland) were obtained using digital calipers [18, 27]. The PY were weighed and photographed, and the developmental features were noted before the PY were returned to the teat and/or the pouch [31].
Milk Analysis
Milk samples from each milking interval were analyzed by reverse-phase high-performance liquid chromatography (HPLC) essentially as described by Simpson et al. [15]. Purified whey (
40 µl) was fractionated on a Poros R2/H reverse-phase column (4.6 x 100 mm; Pharmacia, Sydney, NSW, Australia) using a gradient of 15%–60% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid at a flow rate of 4 ml/min. The absorbance of the eluate was measured at 215 and 280 nm. Protein peaks were collected, dried under vacuum, and stored at 4°C. Samples were reconstituted in appropriate volumes of double-distilled water. All whey samples and HPLC-purified fractions were analyzed by 20% SDS-PAGE [32], and proteins were visualized by staining with Coomassie blue R-250 in 10% methanol/7% (v/v) acetic acid. Milk was diluted in double-distilled water, and the carbohydrate content was determined [9]. The protein content of whole milk was measured using the BCA microtiter plate protein assay (Pierce and Warriner, Sydney, NSW, Australia) using bovine serum albumin standards.
Statistical Analysis
Student t-test was used to determine statistical differences between the two groups over the course of the experiment. Body weights were log transformed to linearize the relationship between weight and age, and then linear regression analysis was performed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To create a 56-day delay in the sucking pattern of tammars, 60-day-old PY were fostered to lactating mothers at fortnightly intervals from Day 60 of lactation (Fig. 1). During this time, the PY were weighed at the beginning (age, 60 days) and end (age, 74 days) of the individual foster periods from Day 60 to 130 of lactation, and the weight gains were calculated (Fig. 2). The foster and control PY gained the same weight during the first 2 wk (60–74 days of lactation) when both groups of PY were the same age (P > 0.1) (Fig. 2). However, the weight gain of each group of 60-day-old, fostered PY suckled by mothers at Days 74–130 of lactation was significantly greater than the 60-day-old, fostered PY suckled by mothers at Days 60–74 of lactation (P < 0.001) (Fig. 2). The fostered PY gained more weight than the control PY between Days 74 and 116 of lactation (P < 0.05) (Fig. 2) but not between Days 116 and 130 of lactation (P > 0.05) (Fig. 2).
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Growth of PY During the Remainder of Lactation (Days 116–289)
Following the final foster period, both sets of PY were weighed at intervals of 2–3 wk until Day 289 of lactation (Fig. 1). The final group of foster PY weighed less than the controls at Day 116 of lactation and remained lighter than the controls through Day 172 (actual foster PY age, 60–116 days; P < 0.05) (Fig. 3A). From Days 193 to 228, no difference was found between the two groups (P > 0.05) (Fig. 3A). From Days 242 to 270, the foster PY body weights again lagged behind those of the controls (P < 0.05), but by Day 289, no significant difference was observed in weight between the groups (P > 0.05) (Fig. 3A). This is demonstrated in Figure 3B, in which the two linear regression lines of foster and control PY log-transformed body weights converge to the same body weight at the end of lactation.
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When compared at the same age, the foster PY weighed more than the control PY at every stage (Fig. 3C). Even though the final group of 60-day-old PY that were fostered weighed more than the 60-day-old control PY (P < 0.05) (Fig. 3C), the foster PY still gained weight more rapidly than the controls. Using linear regression analysis, the growth rate of the foster PY was 2.4% per day, which was significantly higher than the 2% per day growth rate of the controls during the lactation period (P < 0.001) (Fig. 3D). At the final milking, the foster PY were 233 days old, and their weight was 2- to 3-fold more than that of the controls at a similar age (Fig. 3C) but was the same as the weight of 289-day-old control PY (P > 0.05) (Fig. 3, C and D).
Development of Foster and Control PY
A set of developmental features was chosen as objective markers of PY development, and all PY were assessed for these characteristics (Table 1). Development of the controls followed published profiles, with eyes opening at approximately Day 140, standing by Day 160, fur development complete at approximately Day 220, and pouch exit beginning at approximately Day 250 (Table 1) [3]. In contrast, a striking difference between the developmental profiles of the foster and control PY was observed during the interval from Day 150 to 228 of lactation. For example, the foster PY at an actual age of 172 days had a substantial covering of fur and were able to hop, whereas the control PY had a light covering of fur and were only just able to stand on their own (Fig. 4A). Similarly, a 214-day-old foster PY had fur development equivalent to an adult, whereas the control PY had not advanced to the rapid growth phase and were still developing fur and motor coordination (Fig. 4B).
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Growth of Mammary Glands
The MI is a noninvasive measurement of gland size that is determined by multiplying the maximum perpendicular dimensions of the gland [5, 18]. The MI increased during lactation, and no significant differences were found between the host and control tammars from Days 70 to 270 of lactation (P > 0.5) (Fig. 5), even though the host MI peaked approximately 2 wk after the control MI. The control PY were commencing permanent pouch exit at approximately Day 250, and from this point onward, a decline was observed in the MI (Fig. 5) similar to that published by Green [1]. A comparable decline in the hosts was not observed until Day 270 (Fig. 5). Permanent exit from the pouch by the control PY at Day 289 of lactation was indicated by the appearance of dirty pouches, whereas the host tammars at the same stage of lactation had clean pouches (data not shown). The experiment was terminated at Day 289 of lactation, before the foster PY had permanently left the pouch.
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Changes in Milk Carbohydrate and Total Milk Protein Concentrations
The carbohydrate content and total protein concentration was measured in the milk from each host and control animal at regular intervals throughout lactation (Fig. 1). The carbohydrate content of host milk was significantly higher than that in the control group at Days 74 and 151–193 of lactation (P < 0.05) (Fig. 6A). At approximately Day 220 of lactation, the carbohydrate content of milk from control tammars began to decline, whereas in the host animals, this decline occurred approximately 3 wk later (Fig. 6A). This resulted in a significantly different milk carbohydrate concentration at Day 256 of lactation (P < 0.05) (Fig. 6A). Milk samples analyzed from a separate group of tammars that were not originally included in this study (i.e., nonexperimental tammars) showed the decline in carbohydrate concentration occurred significantly earlier compared to the control group of tammars, resulting in different milk carbohydrate concentrations at Day 256 of lactation (P > 0.05) (Fig. 6A). However, the timing of carbohydrate concentration decline in both the control and non-experimental tammars fell within a previously observed range [9].
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No difference was found between the total milk protein concentrations of each experimental group during the lactation period with the exception of Day 270, when the protein content of milk from the host tammars was significantly less (P < 0.05) (Fig. 6B). Furthermore, no difference was observed in the timing of the major increase in milk protein between the host and control and tammars (Fig. 6B). The total protein content in milk from the nonexperimental tammars was lower than in the experimental groups, probably because of different dietary protein levels between the two groups [33], but the rate of increase in protein concentration was similar (Fig. 6B).
Timing of Secretion of ELP and WAP
The transition between phase 2A and phase 2B is marked by the down-regulation of ELP gene expression and the induction of WAP gene expression [15, 16]. The ELP is not easily detectable by SDS-PAGE, and in the absence of antibodies for either ELP or WAP, HPLC was used to accurately determine the timing of changes in the secretory pattern of these proteins. The secretion of ELP in milk from control tammars declined at approximately Day 116 of lactation, approximately 2 wk before that shown in whey from host tammars. This protein was not detected in milk at 130 days of lactation from either treatment group (Fig. 7A). This result was confirmed by SDS-PAGE analysis of the column eluates (data not shown).
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Down-regulation of ELP preceded the appearance of WAP in both the host and control milk samples at approximately 130 days of lactation (Fig. 7B), eluting as either a single or double peak by HPLC [17]. The WAP was detected at high levels at Days 228 and 256 in the hosts and at comparatively low levels in the controls at Day 256 of lactation (Fig. 7B), indicating that a delay occurred in down-regulation of WAP secretion in the host tammars. This result was confirmed by the SDS-PAGE analysis of whey from host and control groups, showing that WAP secretion was extended by approximately 2 wk in the host group of tammars (Fig. 8).
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Timing of Induction of LLP-A and LLP-B Secretion
The SDS-PAGE analysis of whey samples from Days 151 to 289 of lactation showed that LLP-A was first detectable between Days 172 to 214 of lactation, with no difference in the time of induction between host and control tammars (Fig. 8). The secretion of LLP-B was first detectable between Days 193 to 228 of lactation, and as with LLP-A, no difference was observed in the time of induction between host and control tammars (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The timing of either induction or down-regulation of the major phase-specific milk proteins WAP, ELP, LLP-A, and LLP-B was not correlated with the 56-day delay in onset of the sucking pattern on the host tammars, indicating that milk composition was regulated by the maternal program. A brief (
14 days) delay occurred in the down-regulation of WAP and ELP secretion, although this was considerably less than the delay of 56 days in the sucking pattern of the foster PY. The secretion of LLP-A and LLP-B was not affected by the altered sucking stimulus, which is consistent with a previous study showing that synthesis of the LLPs is regulated by the developmental status of the gland (unpublished results). The carbohydrate and protein secretion profiles were similar in the host and control groups with one exception: The decline in carbohydrate concentration was delayed by approximately 2–3 wk in the host group, suggesting a potential contribution from the PY to changes in specific milk components. However, this is far less important than maternal regulation. In general, these findings indicate that the pattern of secretion for milk components follows an endogenous maternal program, which is consistent with previous findings [26, 27].
The developmental status of the young was strongly influenced by the stage of lactation, suggesting that regulation of PY development is significantly influenced by changes in milk composition. Phase 2A milk is high in asparagine residues [11], which may be attributed to the synthesis of ELP [15]. Asparagine has been shown to be critical for brain development in nursing rat young [34], correlating well with the fact that brain development is largely complete by the end of phase 2A [3, 11]. During phase 2B, the sulfur-containing amino acids, cysteine (and/or cystine) and methionine, increase at approximately Day 150, at the time of hair follicle and nail development [11]. This coincides with secretion of the major whey protein in phase 2B, WAP, which is high in cysteine residues [16]. Foster young were 95 days old when they first received milk containing increased sulfur amino acids, and fur development was complete 9 wk later (age, 150 days) instead of after the anticipated 14 wk. Similarly, fur development in control PY was completed 6 wk after receiving milk that contained increased sulfur amino acids (age, 190 days).
Our data confirm that the foster PY were receiving milk of higher energy content and a different protein composition relative to that provided to control PY of the same age. Green et al. [6] calculated that the energy content of milk at Day 7 of lactation is 250 kJ per 100 g and, by Day 176, rises to 500 kJ per 100 g, peaking at approximately Day 246 at 1150 kJ per 100 g. Given that foster PY at an actual age of 137 days were sucking from glands at Day 193 of lactation, their total energy intake would have been increased compared to normal. This could explain the very fat appearance of the foster PY from Days 193 to 228 of lactation and their accelerated growth rate.
The sucking pattern of the foster PY did not appear to influence the size of the mammary gland or the apparent increases in the volume of milk produced. Therefore, the increased growth rates of these animals may also reflect an increase in milk intake. This phenomenon has been observed previously when younger PY fostered onto glands more than 60 days further into lactation became obese [26, 27]. Conversely, PY became emaciated after being transferred to lactating glands that were at least 60 days younger, presumably because of lack of milk production [26, 27]. In contrast, the milking frequency of goats and cows is inversely associated with residual milk volume in the gland, in which increased residual milk volume suppresses milk production, and vice versa [25, 35]. This feedback inhibition of milk synthesis has been attributed to a putative glycoprotein called FIL. In the tammar, the sucking pattern of the young did not appear to affect the level of milk production, indicating that FIL may not play a role in long-term regulation of lactation.
The data presented in this paper provide sound evidence to challenge the original dogma that ongoing lactation is largely regulated by the sucking young. We show that the result of fostering younger animals onto lactating females, effectively retarding the developmental age of the sucking PY, is that the gross milk composition and secretion of specific milk proteins remains essentially unchanged and that the developmental rate of the fostered young is dramatically increased, presumably by receiving additional milk of altered composition. Our findings lead to an alternate hypothesis in which the female tammar has dominant regulation over mammary gland development, milk composition, and PY development, regardless of the age of the PY, and suggest that the sucking pattern does not significantly influence these processes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 61 3 8344 7909; e-mail: k.nicholas{at}zoology.unimelb.edu.au
3 Current address: Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
4 Current address: Department of Biochemistry and Molecular Biology, The University of Melbourne, Victoria 3010, Australia
5 Both authors contributed equally to the research
Received: 25 March 2002.
First decision: 18 April 2002.
Accepted: 24 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) animals throughout lactation. The nonexperimental group consisted of samples from animals that were milked a variable number of times (n =1–6). Each value is the mean ± SEM (n = 3–6) or range (if n = 2, nonexperimental animals only). The stage of lactation corresponds to the PY age of the control animals. A) The average carbohydrate content of milk at each milking time point. B) The average total protein content of milk at each milking time point
