Metoclopramide induces preparturient, low-level hyperprolactinemia to increase milk production in primiparous sows.

Inadequate milk production by sows often limits the growth of piglets. A successful lactation requires prolactin (PRL)-induced differentiation of the alveolar epithelium within the mammary glands of sows between days 90-110 of gestation. We hypothesized that induction of late gestational hyperprolactinemia in primiparous sows by oral administration of the dopamine antagonist metoclopramide (MET) would enhance mammary epithelial differentiation, milk yield, and piglet growth rate and that these effects would carry over into a subsequent lactation. Twenty-six gilts were assigned to receive either MET (n = 13, 0.8 mg/kg) or vehicle (CON, n = 13) twice daily from days 90-110 of gestation. The same sows were followed into their second lactation without additional treatment. On day 90 of gestation, circulating PRL concentrations peaked 45 min after feeding MET (P < 0.001) and then returned to baseline 3 h later. This response occurred daily out to day 104 of gestation (P < 0.05). Compared with CON, MET-treated gilts had enlarged alveoli on gestation day 110 (P < 0.05). Treatment with MET did not affect feed intake, body weight, or body fatness during pregnancy or lactation. Piglets born to MET-treated sows had both increased body weights and average daily gain on lactation days 14 and 21 (P < 0.05). Milk intake by piglets was estimated from deuterium oxide dilution. Although milk intake by piglets nursing MET sows was not statistically different from those nursing CON sows on day 21 of lactation (P = 0.18), there was a greater increase in milk consumption by piglets born to MET-treated sows between days 9 and 21 of lactation than for those in CON litters (P < 0.001). In one group of second parity sows (n = 11) that were treated with MET during their first gestation, milk yield increased by 21% during their second lactation (P < 0.05) in association with a 14% decline in body fatness across lactation compared with a 7% decline in CON sows (P < 0.05). These findings demonstrate that MET-induced hyperprolactinemia in primiparous sows during late pregnancy can increase milk yield and piglet growth rate, setting the stage for further large-scale studies.

Upregulation of MARCKS in kidney cancer and its potential as a therapeutic target.

Targeted therapeutics, such as those abrogating hypoxia inducible factor (HIF)/vascular endothelial growth factor signaling, are initially effective against kidney cancer (or renal cell carcinoma, RCC); however, drug resistance frequently occurs via subsequent activation of alternative pathways. Through genome-scale integrated analysis of the HIF-α network, we identified the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) as a potential target molecule for kidney cancer. In a screen of nephrectomy samples from 56 patients with RCC, we found that MARCKS expression and its phosphorylation are increased and positively correlate with tumor grade. Genetic and pharmacologic suppression of MARCKS in high-grade RCC cell lines in vitro led to a decrease in cell proliferation and migration. We further demonstrated that higher MARCKS expression promotes growth and angiogenesis in vivo in an RCC xenograft tumor. MARCKS acted upstream of the AKT/mTOR pathway, activating HIF-target genes, notably vascular endothelial growth factor-A. Following knockdown of MARCKS in RCC cells, the IC50 of the multikinase inhibitor regorafenib was reduced. Surprisingly, attenuation of MARCKS using the MPS (MARCKS phosphorylation site domain) peptide synergistically interacted with regorafenib treatment and decreased survival of kidney cancer cells through inactivation of AKT and mTOR. Our data suggest a major contribution of MARCKS to kidney cancer growth and provide an alternative therapeutic strategy of improving the efficacy of multikinase inhibitors.

TRIENNIAL LACTATION SYMPOSIUM/BOLFA: Dietary regulation of allometric ductal growth in the mammary glands.

Although mammary gland growth and development in females is a lifelong process, it builds on isometric and allometric phases of mammary growth to establish a complex ductal network before and during puberty. Only then can other phases of branching and alveologenesis, differentiation, lactation, and involution proceed. Although the ductal network of various species differs in its histomorphology, all glands undergo a common phase of allometric growth when the mammary ducts penetrate into the supporting stromal microenvironment. Perhaps not surprisingly, different aspects of diet and nutrition can influence this allometric growth, either directly or indirectly. In this review, we outline some of the fundamental aspects of how allometric ductal growth in the mammary glands of various species is influenced by diet and nutrition and identify opportunities and questions for future investigation.

Regulation and localization of vascular endothelial growth factor within the mammary glands during the transition from late gestation to lactation.

The vascular network within the developing mammary gland (MG) grows in concert with the epithelium to prepare for lactation, although the mechanisms coordinating this vascular development are unresolved. Vascular endothelial growth factor A (VEGF-A) mediates angiogenesis and vascular permeability in the MG during pregnancy and lactation, where its expression is upregulated by prolactin. Given our previous finding that late-gestational hyperprolactinemia induced by domperidone (DOM) increased subsequent milk yield from gilts, we sought to establish changes in vascular development during late gestation and lactation in the MGs of these pigs and determine whether DOM altered MG angiogenesis and the factors regulating it. Gilts received either no treatment (n = 6) or DOM (n = 6) during late gestation, then had their MG biopsied from late gestation through lactation to assess microvessel density, VEGF-A distribution and messenger RNA expression, and aquaporin (AQP) gene expression. Microvessel density in the MG was unchanged during gestation then increased between days 2 and 21 of lactation (P < 0.05). The local expression of messenger RNA for VEGF-A120, VEGF-A147, VEGF-A164, VEGF-A164b, VEGF-A188, VEGF receptors-1 and -2, and AQP1 and AQP3 all generally increased during the transition from gestation to lactation (P < 0.05). Immunostaining localized VEGF-A to the apical cytoplasm of secretory epithelial cells, consistent with a far greater concentration of VEGF-A in colostrum and/or milk vs plasma (P < 0.0001). There was no effect of DOM on any of the variables analyzed. In summary, we found that vascular development in the MG increases during lactation in first-parity gilts and that VEGF-A is a part of the mammary secretome. Although late-gestational hyperprolactinemia increases milk yield, there was no evidence that it altered vascular development.

Neoplastic transformation of porcine mammary epithelial cells in vitro and tumor formation in vivo.

BACKGROUND: The mammary glands of pigs share many functional and morphological similarities with the breasts of humans, raising the potential of their utility for research into the mechanisms underlying normal mammary function and breast carcinogenesis. Here we sought to establish a model for the efficient manipulation and transformation of porcine mammary epithelial cells (pMEC) in vitro and tumor growth in vivo. METHODS: We utilized a vector encoding the red florescent protein tdTomato to transduce populations of pMEC from Yorkshire -Hampshire crossbred female pigs in vitro and in vivo. Populations of primary pMEC were then separated by FACS using markers to distinguish epithelial cells (CD140a-) from stromal cells (CD140a+), with or without further enrichment for basal and luminal progenitor cells (CD49f+). These separated pMEC populations were transduced by lentivirus encoding murine polyomavirus T antigens (Tag) and tdTomato and engrafted to orthotopic or ectopic sites in immunodeficient NOD.Cg-Prkdc (scid) Il2rg (tm1Wjl) /SzJ (NSG) mice. RESULTS: We demonstrated that lentivirus effectively transduces pMEC in vitro and in vivo. We further established that lentivirus can be used for oncogenic-transformation of pMEC ex vivo for generating mammary tumors in vivo. Oncogenic transformation was confirmed in vitro by anchorage-independent growth, increased cell proliferation, and expression of CDKN2A, cyclin A2 and p53 alongside decreased phosphorylation of Rb. Moreover, Tag-transformed CD140a- and CD140a-CD49f + pMECs developed site-specific tumors of differing histopathologies in vivo. CONCLUSIONS: Herein we establish a model for the transduction and oncogenic transformation of pMEC. This is the first report describing a porcine model of mammary epithelial cell tumorigenesis that can be applied to the study of human breast cancers.

Late gestational hyperprolactinemia accelerates mammary epithelial cell differentiation that leads to increased milk yield.

The growth rate of piglets is limited by sow milk yield, which reflects the extent of epithelial growth and differentiation in the mammary glands (MG) during pregnancy. Prolactin (PRL) promotes both the growth and differentiation of the mammary epithelium, where the lactational success of pigs is absolutely dependent on PRL exposure during late gestation. We hypothesized that inducing hyperprolactinemia in primiparous gilts during late gestation by administering the dopamine antagonist domperidone (DOM) would increase MG epithelial cell proliferation and differentiation, subsequent milk yield, and piglet growth. A total of 19 Yorkshire-Hampshire gilts were assigned to receive either no treatment (CON, n = 9) or DOM (n = 10) twice daily from gestation d 90 to 110. Serial blood sampling during the treatment period and subsequent lactation confirmed that plasma PRL concentrations were increased in DOM gilts on gestation d 91 and 96 (P < 0.001). Piglets reared by DOM-treated gilts gained 21% more BW during lactation than controls (P = 0.03) because of increased milk production by these same gilts on d 14 (24%, P = 0.02) and 21 (32%, P < 0.001) of lactation. Milk composition did not differ between the 2 groups on d 1 or 20 of lactation. Alveolar volume within the MG of DOM-treated gilts was increased during the treatment period (P < 0.001), whereas epithelial proliferation was unaffected by treatment. Exposure to DOM during late gestation augmented the postpartum increase in mRNA expression within the MG for ß-casein (P < 0.03), acetyl CoA carboxylase-α (P < 0.01), lipoprotein lipase (P < 0.06), α-lactalbumin (P < 0.08), and glucose transporter 1 (P < 0.06). These findings demonstrate that late gestational hyperprolactinemia enhances lactogenesis within the porcine MG and increases milk production in the subsequent lactation.

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.

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.

Alternative splicing to exon 11 of human prolactin receptor gene results in multiple isoforms including a secreted prolactin-binding protein.

Endocrine and autocrine prolactin (PRL) exerts effects on normal breast and breast cancer cells, and high serum PRL is a poor prognostic factor for colorectal cancer. Here we tested the hypothesis that short isoforms of the PRL receptor (PRLR) in human tissue regulate the actions of PRL in cancer. Using 3' RACE we isolated five splice variants of the human PRLR (hPRLR), three of which encode the complete extracellular binding domain. Two of these isoforms, short form 1a (SF1a) and short form 1b (SF1b), possess unique intracellular domains encoded by splicing to exon 11 from exons 10 and 9 respectively. A third novel isoform (delta7/11) reflects alternative splicing from exon 7 to exon 11 and encodes a secreted soluble PRL-binding protein. Additional splice variants of SF1b and delta7/11 that lacked exon 4 (delta4-SF1b and delta4-delta7/11) were also identified. Functional analyses indicated that hPRLR-SF1b is a strong dominant-negative to the differentiative function of the PRLR long form while hPRLR-SF1a is a weaker dominant-negative. Differential abundance of SF1a, SF1b and delta7/11 expression was detected in normal breast, colon, placenta, kidney, liver, ovary and pancreas, and breast and colon tumors. Taken together, these data indicate the presence of multiple isoforms of the hPRLR that may function to modulate the endocrine and autocrine effects of PRL in normal human tissue and cancer.

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.

Biochemistry of esterases associated with organophosphate resistance in Lucilia cuprina with comparisons to putative orthologues in other Diptera.

Esterase activities associated with organophosphate insecticide resistance in the Australian sheep blowfly, Lucilia cuprina, are compared with similar activities in other Diptera. The enzymes making the major contribution to methyl butyrate hydrolysis ("ali-esterase") in L. cuprina, M. domestica, and D. melanogaster comigrate during electrophoresis. The enzymes in L. cuprina and D. melanogaster correspond to the naphthyl acetate hydrolyzing E3 and EST23 isozymes of those species. These and previously published data suggest that the ali-esterases of all three species are orthologous. Strains of L. cuprina fall into four groups on the basis of quantitative determinations of their ali-estesterase, OP hydrolase, and malathion carboxylesterase activities and these groups correspond to their status with respect to two types of OP resistance. Strains susceptible to OP's have high ali-esterase, low OP hydrolase, and intermediate MCE activities; those resistant to malathion but not diazinon have low ali-esterase, intermediate OP hydrolase, and high MCE activities; those resistant to diazinon but not malathion have low ali-esterase, high OP hydrolase, and low MCE activities; those resistant to both OPs have low ali-esterase, high OP hydrolase, and high MCE activities. The correlated changes among the three biochemical and two resistance phenotypes suggest that they are all properties of one gene/enzyme system; three major allelic variants of that system explain OP susceptibility and the two types of OP resistance. Models are proposed to explain the joint contribution of OP hydrolase and MCE activities to malathion resistance and the invariant association of low ali-esterase and elevated OP hydrolase activities in either type of resistance.