T.B. McFadden

Hormonal regulation of mammary differentiation and milk secretion

The endocrine system coordinates development of the mammary gland with reproductive development and the demand of the offspring for milk. Three categories of hormones are involved. The levels of the reproductive hormones, estrogen, progesterone, placental lactogen, prolactin, and oxytocin, change during reproductive development or function and act directly on the mammary gland to bring about developmental changes or coordinate milk delivery to the offspring. Metabolic hormones, whose main role is to regulate metabolic responses to nutrient intake or stress, often have direct effects on the mammary gland as well. The important hormones in this regard are growth hormone, corticosteroids, thyroid hormone, and insulin. A third category of hormones has recently been recognized, mammary hormones. It currently includes growth hormone, prolactin, PTHrP, and leptin. Because a full-term pregnancy in early life is associated with a reduction in breast carcinogenesis, an understanding of the mechanisms by which these hormones bring about secretory differentiation may offer clues to the prevention of breast cancer.

Onset of lactation in the bovine mammary gland: gene expression profiling indicates a strong inhibition of gene expression in cell proliferation

The mammary gland undergoes dramatic functional and metabolic changes during the transition from late pregnancy to lactation. To better understand the molecular events underlying these changes, we analyzed expression profiles of approximately 23,000 gene transcripts in bovine mammary tissue about day 5 before parturition and day 10 after parturition. At the cutoff criteria of the signed fold change ≥2 or ≤−2 and false discovery rate (FDR) ≤0.1, a total of 389 transcripts (1.6%) were significantly differentially expressed at the two stages. Of these transcripts with significant changes, 105 were up-regulated while 284 were down-regulated. Gene ontology analysis showed that the main up-regulated genes were those associated with transport activity (amino acid, glucose, and ion transporters), lipid and carbohydrate metabolism (lipoprotein lipase, acetyl-Coenzyme A synthetases, 6-phosphofructo-2-kinase, etc.), and cell signaling factors (protein p8, Rab18, etc.). The main down-regulated genes were associated with cell cycle and proliferation (cyclins, cell division cycle associated proteins, etc.), DNA replication and chromosome organization (centromere proteins, minichromosome maintenance proteins, histone, etc.), microtubule-based processes (microtubule associated protein tau, kinesin, tubulins, etc.), and protein and RNA degradation (proteasome, proteasome activator, RNA binding motif protein, etc.). The increased expression of glucose transporter GLUT1 mRNA during lactation was verified by quantitative reverse transcription/polymerase chain reactin (PCR) (P < 0.05). GLUT1 protein also increased twofold during lactation (P < 0.05). Furthermore, GLUT1 protein was primarily localized in mammary ductal epithelia and blood vessel endothelia before parturition, but was predominantly localized in the basolateral and apical membranes of mammary alveolar epithelial cells during lactation. Our microarray data provide insight into the molecular events in the mammary gland at the onset of lactation, indicating the up-regulation of genes involved in milk synthesis concomitant with the inhibition of those related to cell proliferation.

Local IGF-I Axis in Peripubertal Ruminant Mammary Development

The regulation of mammary growth and development in heifers is accomplished by complexinteractions of hormones, growth factors, and extracellular matrix molecules. Many of thesegrowth stimulators are believed to be locally produced in the mammary gland and to beaffected by developmental and nutritional status. Although estrogen and growth hormone areconsidered critical to pubertal mammogenesis, results summarized in this review suggest thatIGF-I6 and IGF binding proteins are especially important locally-produced growth regulatorsin peripubertal ruminants. This assertion is supported by studies of ovariectomized heifers, inwhich increased stromal IGFBP-3 and reduced IGF-I correspond with a failure of udderdevelopment. Similarly, reduced mammary development with overfeeding coincides withreduced mitogenic activity of mammary tissue extracts and altered concentrations of IGF-Iand IGFBPs. In vitro studies convincingly demonstrate that much of the mitogenic activity ofmammary extracts or serum can be attributed to IGF-I and that alterations in IGFBP-3 modulateits effectiveness. Thus by analogy to second messenger mechanisms of action for proteinhormones, local mammary-derived growth factors likely explain many of the effects attributedto the classic mammogenic hormones.

The effects of milk removal or four-times-daily milking on mammary expression of genes involved in the insulin-like growth factor-I axis.

Frequent milking of dairy cows during early lactation elicits both an immediate increase in milk yield and a partial carryover effect that persists to the end of lactation. We hypothesized that the immediate response would be associated with a local increase in insulin-like growth factor (IGF)-I signaling and a consequent increase in mammary growth. Four multiparous cows were assigned at parturition to unilateral frequent milking [UFM; milking of the left udder half twice daily (2x; 0230 and 1430 h); milking of the right udder half 4 times daily (4x; 0230, 0530, 1430, and 1730 h)]. Mammary biopsies were obtained from both udder halves at 5 d in milk at 0530 h (immediately after 4x glands were milked). Incorporation of [3H]-thymidine into DNA and mammary cell apoptosis were not affected by UFM. Because biopsies were obtained when udder halves were at different postmilking intervals, our results reflected both the acute, transient mammary response to milking and the sustained mammary response to frequent milking treatment. We further hypothesized that the acute, transient response involves mechanisms distinct from those regulating the sustained response to frequent milking. To test that hypothesis, mammary biopsies were obtained from UFM cows (n=5) at 0500 h, when time postmilking was the same for both udder halves. Mammary cell apoptosis was not affected by UFM. Expression of genes involved in the IGF-I axis was analyzed to identify acute responses associated with milking, per se, versus sustained responses to frequent milking treatment. Removal of milk from 4x glands was associated with an acute increase in expression of IGF binding protein-1, -3, and -4 mRNA in 2x glands, whereas IGF-I expression was increased by frequent milking treatment. These effects, however, were significant only for expression of IGF binding protein-3. Expression of IGF-I receptor did not differ because of milking frequency but was higher in both udder halves immediately postmilking, indicating a systemic effect. We conclude that several genes of the IGF-I axis respond to milking, per se, or frequent milking treatment, via at least 3 distinct patterns. Increased milking frequency does not alter mammary cell proliferation or apoptosis at 5 d in milk; however, it may increase the bioavailability of IGF-I in the mammary gland. Moreover, the increase in local expression of IGF-I in 4x udder halves indicates a role for this gene in the immediate milk yield response to frequent milking during early lactation.

Acute milk yield response to frequent milking during early lactation is mediated by genes transiently regulated by milk removal

Milking dairy cows four times daily (4×) instead of twice daily (2×) during early lactation stimulates an increase in milk yield that partly persists through late lactation; however, the mechanisms behind this response are unknown. We hypothesized that the acute mammary response to regular milkings would be transient and would involve different genes from those that may be specifically regulated in response to 4×. Nine multiparous cows were assigned at parturition to unilateral frequent milking (UFM; 2× of the left udder half, 4× of the right udder half). Mammary biopsies were obtained from both rear quarters at 5 days in milk (DIM), immediately after 4× glands had been milked (experiment 1, n = 4 cows), or 2.5 h after both udder halves had last been milked (experiment 2, n = 5 cows). Affymetrix GeneChip Bovine Genome Arrays were used to measure gene expression. We found 855 genes were differentially expressed in mammary tissue between 2× vs. 4× glands of cows in experiment 1 (false discovery rate ≤ 0.05), whereas none were differentially expressed in experiment 2 using the same criterion. We conclude that there is an acute transcriptional response to milk removal, but 4× milking did not elicit differential expression of unique genes. Therefore, there does not appear to be a sustained transcriptional response to 4× milking on day 5 of lactation. Using a differential expression plot of data from both experiments, as well as qRT-PCR, we identified at least two genes (chitinase 3-like-1 and low-density lipoprotein-related protein-2 that may be responsive to both milk removal and to 4× milking. Therefore, the milk yield response to 4× milking may be mediated by genes that are acutely regulated by removal of milk from the mammary gland.

Regulation of Mammary Development as It Relates to Changes in Milk Production Efficiency

© 2012 Wall and McFadden, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Regulation of Mammary Development as It Relates to Changes in Milk Production Efficiency

Milk Removal: The Key to Maintaining Milk Production and a Tool to Enhance Efficiency

© 2012 Wall and McFadden, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Milk Removal: The Key to Maintaining Milk Production and a Tool to Enhance Efficiency