High-fat diet (HFD) during lactation alters milk composition and is associated with development of metabolic diseases in the offspring. We hypothesized that HFD affects milk microRNA (miRNA) and mRNA content, which potentially impact offspring development. Our objective was to determine the effect of maternal HFD on secreted milk transcriptome. To meet this objective, 4 wk old female ICR mice were divided into two treatments: control diet containing 10% kcal fat and HFD containing 60% kcal fat. After 4 wk on CD or HFD, mice were bred while continuously fed the same diets. On postnatal day 2 (P2), litters were normalized to 10 pups, and half the pups in each litter were cross-fostered between treatments. Milk was collected from dams on P10 and P12. Total RNA was isolated from milk fat fraction of P10 samples and used for mRNA-Seq and small RNA-Seq. P12 milk was used to determine macronutrient composition. After 4 wk of prepregnancy feeding HFD mice weighed significantly more than did the control mice. Lactose and fat concentration were significantly ( P < 0.05) higher in milk of HFD dams. Pup weight was significantly greater ( P < 0.05) in groups suckled by HFD vs. control dams. There were 25 miRNA and over 1,500 mRNA differentially expressed (DE) in milk of HFD vs. control dams. DE mRNA and target genes of DE miRNA enriched categories that were primarily related to multicellular organismal development. Maternal HFD impacts mRNA and miRNA content of milk, if bioactive nucleic acids are absorbed by neonate differences may affect development.

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63 qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland.

Maintaining metabolic balance is a key factor in the health of dairy cattle during the transition from pregnancy to lactation. Little is known regarding the role of the circadian timing system in the regulation of physiological changes during the transition period. We hypothesized that disruption of the cow's circadian timing system by exposure to chronic light-dark phase shifts during the prepartum period would negatively affect the regulation of homeostasis and cause metabolic disturbances, leading to reduced milk production in the subsequent lactation. The objective was to determine the effect of exposure to chronic light-dark phase shift during the last 5 wk prepartum of the nonlactating dry period on core body temperature, melatonin, blood glucose, β-hydroxybutyric acid (BHB) and nonesterified fatty acid (NEFA) concentrations, and milk production. Multiparous cows were moved to tiestalls at 5 wk before expected calving and assigned to control (CTR; n = 16) or phase-shifted (PS; n = 16) treatments. Control cows were exposed to 16 h of light and 8 h of dark. Phase-shifted cows were exposed to the same photoperiod; however, the light-dark cycle was shifted 6 h every 3 d until parturition. Resting behavior and feed intake were recorded daily. Core body temperature was recorded vaginally for 48 h at 23 and 9 d before expected calving using calibrated data loggers. Blood concentrations of melatonin, glucose, BHB, and NEFA were measured during the pre-and postpartum periods. Milk yield and composition were measured through 60 DIM. Treatment did not affect feed intake or body condition. Cosine fit analysis of 24-h core body temperature and circulating melatonin indicated attenuation of circadian rhythms in the PS treatment compared with the CTR treatment. Phase-shifted cows had lower rest consolidation, as indicated by more total resting time, but shorter resting period durations. Phase-shifted cows had lower blood glucose concentration compared with CTR cows (4 mg/mL decrease), but BHB and NEFA concentrations were similar between PS and CTR cows. Milk yield and milk fat yield were greater in PS compared with CTR cows (2.8 kg/d increase). Thus, exposure to chronic light-dark phase shifts during the prepartum period attenuated circadian rhythms of core body temperature, melatonin, and rest-activity behavior and was associated with increased milk fat and milk yield in the postpartum period despite decreased blood glucose pre-and postpartum. Therefore, less variation in central circadian rhythms may create a more constant milieu that supports the onset of lactogenesis.

The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.

Background. Metabolic and hormonal disturbances are associated with sleep disturbances and delayed onset of lactogenesis II. Research Aims. The aim of this study was to measure sleep using wrist actigraphy during gestation weeks 22 and 32 to determine if sleep characteristics were associated with blood glucose, body mass index, gestational related disease, delayed onset of lactogenesis II, or work schedule. Methods. Demographic data were collected at study intake from primiparous women who wore a wrist actigraph during gestation weeks 22 (n = SO) and 32 (n = 44). Start and end sleep time, total nighttime sleep, sleep efficiency, wake after sleep onset, and sleep fragmentation were measured. Night to night variability was assessed with the root mean square of successive difference. Blood glucose levels, body mass index, and gestational disease data were abstracted from medical charts. Timing of lactogenesis II was determined by survey. Results. Between gestation week 22 and 32, sleep efficiency decreased and fragmentation increased (p < .05). During gestation week 32, blood glucose was negatively correlated with sleep duration, and positively related to fragmentation (p < .OS). Women who experienced delayed lactogenesis II had lower sleep efficiency and greater fragmentation (p < .05), and greater night-to-night variability in sleep start and end time, efficiency, and duration during gestation week 32 (p < .OS). Conclusion. Women with better sleep efficiency and more stable nightly sleep time are less likely to experience delayed onset of lactogenesis II. Interventions to improve sleep may improve maternal health and breastfeeding adequacy.