Josephine F. Trott

The tammar wallaby : A model to study putative autocrine-induced changes in milk composition

The marsupial newborn is immature and the mother has the capacity to alter milk composition significantly during lactation, presumably to meet the nutritional requirements of the developing young. Furthermore, macropodid marsupials may practice asynchronous concurrent lactation (ACL)7whereby the mother provides milk which differs in all the major components from adjacent mammary glands for two young of different ages. This phenomenon suggests that local regulation of mammary function, in addition to endocrine stimuli, is likely to be important for controlling milk composition. This paper explores the possibility that changes in sucking patterns of the young represent the first step in a mechanism to signal the mammary gland for putative autocrine-induced changes in milk composition.

Transcriptional and spatiotemporal regulation of prolactin receptor mRNA and cooperativity with progesterone receptor function during ductal branch growth in the mammary gland

Ductal branching within the mammary gland is stimulated by prolactin (PRL) and progesterone (P) acting through their receptors (PRLR and PR). Analysis of mammary gland PRLR expression revealed increasing expression of the long form (L‐PRLR) and two of the three short forms (S1‐ and S3‐PRLR) during puberty that became maximal late in pubescence and early gestation, then declined during gestation. By contrast, S2‐PRLR mRNA levels remained constant. Examination of stromal PRLR revealed the consistent expression of L‐PRLR mRNA. By contrast, S1‐PRLR was present only in the mammary fat pad of neonates, whereas high neonatal expression of S2‐PRLR became undetectable during puberty. Stromal expression of S3‐PRLR decreased to low levels during puberty and was undetectable during lactation and involution. Exogenous PRL stimulated DNA synthesis in both epithelial and adjacent stromal cells in vivo. Distribution of PRLR mRNA in mammary epithelium was homogeneous before puberty and heterogeneous during puberty, gestation, and early lactation. A mutual role for PRLR and PR was suggested wherein PR mRNA increased beyond 6 weeks to maximal levels during puberty and gestation then became undetectable during lactation. In situ hybridization revealed that PR mRNA distribution is homogeneous in the ductal epithelium before 6 weeks and heterogenous during puberty and gestation and that PRLR and PR are similarly distributed in the ductal epithelium. Neither hormone stimulated DNA synthesis in mammary glands of ovariectomized females while their effects interacted markedly. These results demonstrate differential PRLR transcription by epithelial and stromal cells and a similar distribution of PRLR and PR that may facilitate the interaction between P and PRL during ductal branching in the mammary gland.

Maternal Regulation of Milk Composition, Milk Production, and Pouch Young Development During Lactation in the Tammar Wallaby (Macropus eugenii )

Abstract Specific changes in milk composition during lactation in the tammar wallaby (Macropus eugenii) were correlated with the ages of the developing pouch young (PY). The present experiment was designed to test the hypothesis that the sucking pattern of the PY determines the course of mammary development in the tammar wallaby. To test this hypothesis, groups of 60-day-old PY were fostered repeatedly onto one group of host mothers so that a constant sucking stimulus on the mammary gland was maintained for 56 days to allow the lactational stage to progress 42 days ahead of the age of the young. Analysis of the milk in fostered and control groups showed the timing of changes in the concentration of protein and carbohydrate were essentially unaffected by altering the sucking regime. The only change in milk protein secretion was a small delay in the timing of down-regulation of the secretion of whey acidic protein and early lactation protein in the host tammars. In addition, the rates of growth and development of the foster PY were significantly increased relative to those of the control PY because of ingesting more milk with a higher energy content and different composition than normal for their age. The present study demonstrates that the lactating tammar wallaby regulates both milk composition and the rate of milk production and that these determine the rates of PY growth and development, irrespective of the age of the PY.

Morphogenesis of Mammary Gland Development

Development of the mammary gland in females is a dynamic, orchestrated process that occurs throughout postnatal development. Initiated during embryogenesis, epithelial cells advance into the underlying stromal matrix to form a primitive rudimentary structure. With the onset of puberty this anlage then responds to hormonal and local cues to rapidly establish a ductal network. Whereas in mice this network is relatively simple, in humans there is significantly more branching morphogenesis to develop terminal duct lobular unit structures. With the onset of pregnancy and associated changes in the hormonal and local environment, alveolar development progresses to establish a gland that is densely filled with alveolar structures by the end of pregnancy. Concomitantly, mammary epithelial cells within the gland begin to attain their unique ability to synthesize various milk constituents, such that by parturition, functional lactogenesis can be realized.

Expression of novel lipocalin-like milk protein gene is developmentally-regulated during lactation in the tammar wallaby, Macropus eugenii

We have identified a novel whey protein (late lactation protein B; LLPB) that is first secreted in the milk of the tammar wallaby around day 200 of lactation. The LLPB cDNA clone of 843 base pairs encodes a mature protein of 156 amino acids. LLPB shares 65 and 48% nucleotide and deduced amino acid identity, respectively, with a previously identified late lactation protein A (LLPA). Both these proteins share significant amino acid sequence homology with the lipocalin protein family. Expression of the LLPB gene is induced between days 200 and 240 of lactation, in contrast to expression of the LLPA gene, which is induced at around 145 days of lactation. Maximal expression of both genes in mammary explants from tammars at 213 days of lactation required a combination of prolactin, insulin and hydrocortisone. Transcripts of LLPA, LLPB and beta-lactoglobulin (TBLG) were localized to the same cells by in situ hybridization. A substantial level of alveolar maturation is required for expression of the LLP genes, unlike TBLG, which is expressed in immature alveoli. We hypothesize that the temporal expression of the LLPB and LLPA genes may be regulated both by endocrine stimuli and factors intrinsic to the mammary gland.

Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus)

Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of alpha-lactalbumin, beta-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young.

Tissue-specific regulation of porcine prolactin receptor expression by estrogen, progesterone, and prolactin

Prolactin (PRL) acts through its receptor (PRLR) via both endocrine and local paracrine/autocrine pathways to regulate biological processes including reproduction and lactation. We analyzed the tissue- and stage of gestation-specific regulation of PRL and PRLR expression in various tissues of pigs. Abundance of pPRLR-long form (LF) mRNA increased in the mammary gland and endometrium during gestation while in other tissues it remained constant. There was a parallel increase in the abundance of the pPRLR-LF protein in the mammary gland and endometrium during gestation. We determined the hormonal regulation of pPRLR-LF mRNA expression in various tissues from ovariectomized, hypoprolactinemic gilts given combinations of the replacement hormones estrogen (E(2)), progestin (P), and/or haloperidol-induced PRL. Abundance of pPRLR-LF mRNA in kidney and liver was unaffected by hormone treatments. Expression of uterine pPRLR-LF mRNA was induced by E(2) whereas the effect of E(2) was abolished by co-administering P. The expression of pPRLR-LF mRNA in the mammary gland stroma was induced by PRL, whereas E(2) induced its expression in the epithelium. In contrast to these changes in pPRLR expression, pPRL expression was relatively constant and low during gestation in all tissues except the pituitary. Taken together, these data reveal that specific combinations of E(2), P, and PRL differentially regulate pPRLR-LF expression in the endometrium and mammary glands, and that the action of PRL on its target tissues is dependent upon pPRLR-LF abundance more so than the local PRL expression.

TRIENNIAL LACTATION SYMPOSIUM: Prolactin: The multifaceted potentiator of mammary growth and function

At face value there are clear and established roles for prolactin (PRL) in the regulation of mammary gland growth, lactogenesis, and galactopoiesis. These actions of PRL do not occur in isolation; rather, they are finely attuned to and coordinated with many local, reproductive, and metabolic events in the female. Hence, to understand PRL action at the level of the mammary gland is to understand the systemic and local contexts in which it acts and functions. Herein we review the functions of PRL, its receptors, and the pathways leading to the phenotypes it evokes within the mammary glands, including growth and lactation, across a variety of species. At one level, the actions of PRL are mediated by several PRL receptor (PRLR) isoforms, including its long form and various short PRLR variants that are generated by alternative splicing in a species- and tissue-dependent manner. In turn, these PRLR activate a variety of intracellular signaling cascades. We also focus on how PRL coordinates with other endocrine cues to impart its effects on the mammary glands, where the ovarian hormones can independently and substantially modulate PRL action. Many of these effects of PRL are also realized at the local level of the mammary gland, either through the autocrine or paracrine synthesis of a multitude of molecules and transcription factors or through its effects on adjacent supporting tissues, including the mammary vasculature. Taken together, it is clear that PRL directs a variety of mechanisms during growth and function of the mammary gland and is deserving of its classification as the master hormone.

Diet-induced metabolic change induces estrogen-independent allometric mammary growth

Lifetime breast cancer risk reflects an unresolved combination of early life factors including diet, body mass index, metabolic syndrome, obesity, and age at first menses. In parallel, the onset of allometric growth by the mammary glands around puberty is widely held to be estrogen (E)-dependent. Here we report that several physiological changes associated with metabolic syndrome in response to a diet supplemented with the trans-10, cis-12 isomer of conjugated linoleic acid lead to ovary-independent allometric growth of the mammary ducts. The E-independence of this diet-induced growth was highlighted by the fact that it occurred both in male mice and with pharmacological inhibition of either E receptor function or E biosynthesis. Reversal of the metabolic phenotype with the peroxisome proliferator-activated receptor-γ agonist rosiglitazone abrogated diet-induced mammary growth. A role for hyperinsulinemia and increased insulin-like growth factor-I receptor (IGF-IR) expression during mammary growth induced by the trans-10, cis-12 isomer of conjugated linoleic acid was confirmed by its reversal upon pharmacological inhibition of IGF-IR function. Diet-stimulated ductal growth also increased mammary tumorigenesis in ovariectomized polyomavirus middle T-antigen mice. Our data demonstrate that diet-induced metabolic dysregulation, independently of ovarian function, stimulates allometric growth within the mammary glands via an IGF-IR-dependent mechanism.

Hormone interactions confer specific proliferative and histomorphogenic responses in the porcine mammary gland

Mammary gland growth and morphogenesis are regulated by interactions between hormones as much as by their individual actions. The effect of these interactions on the mammary gland phenotype in species other than rodents is relatively undefined. We investigated the individual and combined effects of estrogen (E), progestin (P), and prolactin (PRL) on mammary gland development in gilts. Pigs were shown to have a ductal-lobular parenchyma that underwent hormone-stimulated progression of terminal ductal lobular unit (TDLU) morphogenesis similar to that in the human breast. Ovariectomy plus hypoprolactinemia abolished mammary gland growth. Estrogen alone stimulated mammary epithelial cell proliferation, terminal bud formation, and the progression of TDLU1 structures to a TDLU2 morphotype. Maximal epithelial cell proliferation, DNA content, parenchymal area, and morphological development of the porcine mammary gland were realized following treatment with E+PRL or E+P+PRL. In contrast, P alone did not promote epithelial cell proliferation, TDLU type progression, mammary gland growth, or morphogenesis. These data indicate that interactions between E and PRL are the main determinants of growth and morphogenesis in the porcine mammary gland.