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The main component of mammalian maternal care is milk production. The composition of milk has been hypothesized to determine offspring survival, and to vary with offspring sex. Few studies, however, have examined variation in milk composition of wild mammals in seasonal environments, where environmental conditions can impact the ability of mothers to provide care. We investigated individual differences in milk composition and offspring survival in wild eastern grey kangaroos (Macropus giganteus). We analyzed total protein and lipid concentrations in 103 milk samples from 91 females over 2 years. In a year of low forage production, few females that lactated were in poorer condition and produced milk of lower energy content compared to females lactating in a year of high forage production. Females nursing in late winter produced milk with more lipids compared to females at the same stage of lactation in late spring, whose milk had a higher proportion of protein. The milk of larger females contained more protein than lipid, and females in better condition allocated higher proportions of protein to sons than to daughters. Increased protein concentration in milk was correlated with offspring longevity, and this effect was stronger for sons than daughters. A seasonally modulated lactation strategy enables the adjustment of milk composition to available resources. Condition-specific protein allocation in favor of sons for mothers of higher caring ability suggests adaptive sex-biased maternal care in this highly sexually dimorphic mammal.

We report the first isolation and sequencing of genomic BAC clones containing the marsupial milk protein genes Whey Acidic Protein (WAP) and Early Lactation Protein (ELP). The stripe-faced dunnart WAPgene sequence contained five exons, the middle three of which code for the WAPmotifs and four disulphide core domains which characterize WAP. The dunnart ELPgene sequence contained three exons encoding a protein with a Kunitz motif common to serine protease inhibitors. Fluorescence in situ hybridization located the WAPgene to chromosome 1p in the stripe-faced dunnart, and the ELPgene to 2q. Northern blot analysis of lactating mammary tissue of the closely related fat-tailed dunnart has shown asynchronous expression of these milk protein genes. ELPwas expressed at only the earlier phase of lactation and WAPonly at the later phase of lactation, in contrast to beta-lactoglobulin (BLG) and alpha-lactalbumin (ALA) genes, which were expressed in both phases of lactation. This asynchronous expression during the lactation cycle in the fat-tailed dunnart is similar to other marsupials and it probably represents a pattern that is ancestral to Australian marsupials.

Comparing globin genes and their flanking sequences across many species has allowed globin gene evolution to be reconstructed in great detail. Marsupial globin sequences have proved to be of exceptional significance. A previous finding of a beta(beta)-like omega(omega) gene in the alpha(alpha) cluster in the tammar wallaby suggested that the alpha and beta cluster evolved via genome duplication and loss rather than tandem duplication. To confirm and extend this important finding we isolated and sequenced BACs containing the alpha and beta loci from the distantly related Australian marsupial Sminthopsis macroura. We report that the alpha gene lies in the same BAC as the beta-like omega gene, implying that the alpha-omega juxtaposition is likely to be conserved in all marsupials. The LUC7L gene was found 3' of the S. macroura alpha locus, a gene order shared with humans but not mouse, chicken or fugu. Sequencing a BAC contig that contained the S. macroura beta globin and epsilon globin loci showed that the globin cluster is flanked by olfactory genes, demonstrating a gene arrangement conserved for over 180 MY. Analysis of the region 5' to the S. macroura epsilon (epsilon) globin gene revealed a region similar to the eutherian LCR, containing sequences and potential transcription factor binding sites with homology to eutherian hypersensitive sites 1 to 5. FISH mapping of BACs containing S. macroura alpha and beta globin genes located the beta globin cluster on chromosome 3q and the alpha locus close to the centromere on 1q, resolving contradictory map locations obtained by previous radioactive in situ hybridization.

Homogeneous rat alpha-lactalbumin was prepared from whey by chromatography on DEAE-Sephadex A-50 and Ultrogel AcA 44. Two biologically active forms of alpha-lactalbumin were apparent after ion-exchange chromatography, but on gel filtration the combined forms were eluted as a single peak with a molecular weight of approx. 33000. The molecular weight when determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was 15100. Antiserum to alpha-lactalbumin was prepared from rabbits, and single radial immunodiffusion was used to measure the concentration of alpha-lactalbumin in milk expressed from rats during lactation and for 2 days after the cessation of lactation. A significant positive correlation (r = + 0.89) between the concentrations of alpha-lactalbumin and lactose was obtained for the first 20 days of lactation. This is consistent with the suggestion that alpha-lactalbumin may control the concentration of lactose in milk. However, a significant negative correlation (r = -0.91) between the concentration of alpha-lactalbumin and lactose was obtained for 2 days after the cessation of lactation on day 20.

In the presence of cortisol and prolactin, insulin at concentrations as low as 1 ng/ml significantly stimulates casein synthesis in mammary explants from midpregnant mice; maximal synthesis occurs at 10 ng/ml. However, in the absence of insulin, no detectable immunoprecipitable casein is produced. Insulin also supports enhanced accumulation of casein mRNA in the presence of cortisol and prolactin; neither epidermal growth factor nor somatomedin C has this effect. These inductive actions of insulin are not secondary to a general maintenance effect on the mammary epithelial cell; insulin, epidermal growth factor, and somatomedin C can support the accumulation of RNA in rough endoplasmic reticulum equally well. In addition, these effects do not reflect a specific insulin requirement for prolactin sensitivity; epidermal growth factor can support prolactin-induced total RNA synthesis as well as insulin can. The results demonstrate that, although insulin, epidermal growth factor, and somatomedin C can all function as cell maintenance agents, only insulin, together with cortisol and prolactin, can induce casein mRNA accumulation.

The lactational strategy adopted by marsupials is very different to that of eutherian mammals, in terms of the developmental changes in the composition of the milk during lactation and the hormonal profile associated with these changes. Most studies have focused on the dramatic changes in carbohydrate and fat in the milk with surprisingly little attention being directed to the control of the milk proteins. Studies presented in this chapter have focused on the mechanism of control of two critical stages of the lactation cycle in Macropus eugenii (see Fig.1.1); firstly, the transition from Phase 1 to Phase 2 as lactation is initiated (lactogenesis) and secondly, the transition from Phase 2 to Phase 3 with the accompanying changes in the qualitative and quantitative synthesis of milk These questions have been addressed using a mammary gland explant culture system in an attempt to equate what is known about the hormonal changes in the peripheral circulation at these times with the capacity of the mammary gland to synthesize specific milk proteins. In addition, and just as importantly, the value of the mammary gland of M. eugenii as an unique experimental model to assess questions of fundamental importance in biology will be discussed.

A major whey protein which appears in milk from the tammar wallaby (Macropus eugenii) only during the second half of lactation (late lactation protein-A, LLP-A) was purified to apparent homogeneity by ion-exchange chromatography and gel filtration. An Mr of 21,600 +/- 2000 was calculated from its amino acid composition. A computer-based comparison of the sequence of the first 69 amino acid residues with the Atlas of Protein Sequence data base showed no significant homology with known proteins. Antiserum to LLP-A was prepared in rabbits, and single radial immunodiffusion was used to measure the amounts of LLP-A in milk during the first 40 weeks of lactation. LLP-A was first detected at 26 weeks; thereafter its concentration increased abruptly, to reach a maximum of 26 g/l at approx. 36 weeks of lactation. Explants prepared from mammary gland biopsies at 20 and 35 weeks of lactation were exposed to [3H]amino acids for 8 h; immunoprecipitation of tissue extracts showed that, whereas the rate of casein synthesis was the same at both stages of lactation, LLP-A was synthesized only by the 35-week mammary gland.

The minimal hormonal requirements for the in-vitro accumulation of α-lactalbumin have been investigated in a marsupial, the tammar (Macropus eugenii). Mammary gland explants from 24-day pregnant tammars cultured in medium containing bovine insulin, cortisol and ovine prolactin showed a progressive increase in accumulation of α-lactalbumin during 4 days of incubation. No increment was observed if prolactin was omitted from the medium. However, a similar rate of increase was observed after 3 days of culture in medium containing prolactin alone. This induction of α-lactalbumin was maximal at a prolactin concentration of approximately 0·02 mg/l, which corresponds to physiological levels during pregnancy and early lactation. The absence of an effect of bovine insulin on tammar explants is not due to a general unresponsiveness to this hormone since insulin-stimulated DNA synthesis and amino acid uptake was evident after 3 days of culture. The inclusion of tri-iodothyronine and raised concentrations of cortisol in culture media have been shown to modulate α-lactalbumin synthesis in eutherian mammals but were without effect in the tammar. In addition, increased levels of progesterone did not inhibit the induction of α-lactalbumin, confirming an earlier invivo study suggesting that progesterone withdrawal may not be the lactogenic trigger in this species. Thus the pregnant tammar is the only species yet described in which α-lactalbumin is induced maximally in vitro in response to a single hormone. J. Endocr. (1985) 106, 337–342

Commercial preparations of serum albumin from six species can markedly enhance the prolactin-independent induction of α-lactalbumin in mammary explants from pregnant rats, and evoke such induction in the tissue from virgin rats. These effects are similar to those of epidermal growth factor (EGF) reported previously. The stimulatory activity of bovine serum albumin (BSA) resides in a putative impurity in the albumin. Charcoal extraction and gel filtration of the BSA results in complete loss of activity. Of the five milk protein mRNAs studied, only α-lactalbumin mRNA is induced by insulin, glucocorticoid and serum albumin in the absence of prolactin. Despite these similarities, the biological effects of serum albumin and EGF on mammary tissue diverge in some respects. They appear to operate by different mechanisms since their effects on the rat system are additive. Furthermore, while both inhibit prolactin-mediated induction of α-lactalbumin in rabbit mammary explants, cortisol converts EGF into a stimulatory agent, but merely blocks the inhibitory effect of serum albumin. The results emphasize that commercial serum albumin is not to be regarded simply as an inert protein additive to culture media. J. Endocr. (1988) 119, 133–139