The most common reason for premature breastfeeding cessation is real, or perceived, low milk supply. Maternal stress suppresses milk production, but the mechanism by which stress inhibits lactation has not been defined. The mammalian stress response is mediated in large part by glucocorticoids (GC). It is well-established that excessive endogenous and exogenous GCs transiently suppress milk volume and milk lactose content, without affecting milk fat or protein levels. In vitro studies from the 1980s suggested that the biphasic regulation of alpha-lactalbumin (LALBA) synthesis by GCs is central to the pathophysiology of stress-induced suppression of milk lactose content and milk yield. The LALBA protein is unique to the mammary gland and is required for lactose synthesis and milk production, yet its transcriptional regulation has yet to be defined.

We first sought to define the effect of a synthetic GC (dexamethasone, DEX) on milk yield and composition alongside changes in mammary gene expression in dairy cows. We demonstrated that a single, high dose of DEX administered to lactating dairy cows transiently suppressed milk volume, milk lactose content, milk LALBA content, and LALBA gene expression.

Then, we wanted to determine the reliability and replicability of the murine mammary explant system for the study of GC-mediated regulation of Lalba expression. We defined doses of corticosterone (CORT) and DEX and time intervals at which Lalba expression could be maximally upregulated, inhibited, or suppressed following initial stimulation. We demonstrated for the first time that Lalba expression in mammary explants from midpregnant mice can be suppressed by over 50% in response to high dose of CORT after initial 48 h of stimulation by a low dose of CORT.

The extent to which a decrease in LALBA gene and protein expression contributes to the decline in milk lactose content and milk yield remains to be determined. Future research endeavors should focus on defining the GC-regulated transcriptional landscape at the Lalba promoter using the murine mammary explant system. Determining the mechanism by which GCs modulate LALBA transcription will inform preventative and therapeutic strategies for low milk supply and improve maternal-child health outcomes.

The cell-specific actions for prolactin (PRL) are dependent on the coordinated transcriptional regulation of the prolactin receptor (PRLR) gene across tissues. Several lines of evidence indicate that the use of multiple promoters underlies the tissue specific regulation for PRLR expression in states such as pregnancy and lactation. However, the role and regulation of PRL/PRLR action in the kidneys is less clear. We previously identified that one of several first exons of the porcine PRLR is expressed exclusively in the kidneys, small intestine, and liver. We found the 5’ non-coding region of this first exon (E1.3) is conserved across mammalian PRLR, including ungulates and humans, and its expression increased throughout gestation. We hypothesized E1.3 expression in the kidney was regulated by tissue-specific factors acting on the E1.3 promoter (promE1.3), and its expression increased in support of osmoregulation. Our objectives were to 1) resolve the factor(s) regulating E1.3 expression in the kidneys of pigs and 2) develop an ex vivo model to study E1.3 regulation in the kidneys. We found promE1.3 activation was tissue and cell line specific, whereby the proximal promoter was only active in kidney (LLC-PK1) and colorectal (Caco-2) cells. We also found an element downstream of the transcription start site (TSS) that acts to repress promoter activation in all cell lines tested. In silico transcription factor binding analysis of the proximal promoter revealed multiple consensus sequences for tissue-specific transcription factors, including hepatocyte nuclear factor 1 (HNF1). Mutation of one of the binding sites for HNF1 within the E1.3 promoter resulted in a significant (p<0.05) downregulation in promoter activity in Caco-2 cells, and a reduction in promoter activity in LLC-PK1 cells. Using our kidney explant model, we found E1.3 was more highly expressed than the ubiquitous first exon of the PRLR (exon 1A, E1A) ex vivo. Interestingly E1.3 expression significantly (p<0.05) declined with time in culture, and E1A became the predominant transcript expressed after 48 hours (h). There was no significant difference in PRLR long form or short form expression over time in culture. However, mRNA expression for HNF1 and HNF4 variants declined (p<0.05) over 48h in culture. Together, these data demonstrate that elements within the proximal promoter region of promE1.3 are vital for E1.3 expression, and tissue-specific elements may be required for its sustained expression. It remains to be determined if HNF transcription factors are directly or indirectly responsible for the regulation of PRLR expression in the kidneys of pigs.

The mammary gland (MG) is a remarkable tissue that undergoes a number of stages ofepithelial growth and differentiation and is regulated by hormones such as prolactin (PRL). Recent research has found that binucleated cells (BNC) in the MG of mice form at the time of late gestation and remain until the termination of lactation, although their functional purpose is unknown. In order to improve the understanding of their development pattern, we aimed to quantify the abundance of BNC in the MG of primiparous pigs (n=4) from late gestation day 90 through lactation day 21. Further, we used hyperprolactinemic primiparous gilts (n=4) treated with the dopamine antagonist domperidone (DOM) to test the latter’s effect on the abundance of BNC within the MG during the same period. The cellular architecture was assessed by three-dimensional confocal microscopy and immunofluorescent staining. Our results indicate that BNC abundance increased during the gestation-lactation window to reach maximal levels on day 21 of lactation. We also found that DOM-treated gilts had a significantly greater incidence of BNC compared to controls (p < .05) and that on lactation day 21 there was a statistically greater number of BNCs in DOM-treated sows (p < .05). These findings demonstrate that not only do BNCs exist in the porcine MG but, that they may play a functional role during lactogenesis.