Emerging evidence of the physiological role of hypoxia in mammary development and lactation

Hypoxia is a physiological or pathological condition of a deficiency of oxygen supply in the body as a whole or within a tissue. During hypoxia, tissues undergo a series of physiological responses to defend themselves against a low oxygen supply, including increased angiogenesis, erythropoiesis, and glucose uptake. The effects of hypoxia are mainly mediated by hypoxia-inducible factor 1 （HIF-1）, which is a heterodimeric transcription factor consisting of o and 13 subunits. HIF-1β is constantly expressed, whereas HIF-1α is degraded under normal oxygen conditions. Hypoxia stabilizes HIF-1α and the HIF complex, and HIF then translocates into the nucleus to initiate the expression of target genes. Hypoxia has been extensively studied for its role in promoting tumor progression, and emerging evidence also indicates that hypoxia may play important roles in physiological processes, including mammary development and lactation. The mammary gland exhibits an increasing metabolic rate from pregnancy to lactation to support mammary growth, lactogenesis, and lactation. This process requires increasing amounts of oxygen consumption and results in localized chronic hypoxia as confirmed by the binding of the hypoxia marker pimonidazole HCI in mouse mammary gland. We hypothesized that this hypoxic condition promotes mammary development and lactation, a hypothesis that is supported by the following several lines of evidence： i） Mice with an HIF-1α deletion selective for the mammary gland have impaired mammary differentiation and lipid secretion, resulting in lactation failure and striking changes in milk compositions; ii） We recently observed that hypoxia significantly induces HIF-1α- dependent glucose uptake and GLUT1 expression in mammary epithelial cells, which may be responsible for the dramatic increases in glucose uptake and GLUT1 expression in the mammary gland during the transition period from late pregnancy to early lactation; and iii） Hypoxia and HIF-1α increase the phosphorylation of signal transducers and activators of transcription 5a （STAT5a）in mammary epithelial cells, whereas STATS phosphorylation plays important roles in the regulation of milk protein gene expression and mammary development. Based on these observations, hypoxia effects emerge as a new frontier for studying the regulation of mammary development and lactation.

Influences of lauric acid addition on performance,nutrient digestibility and proteins related to mammary gland development in dairy cows

Lauric acid(LA)has the possibility to improve milk production in dairy cows by improving mammary gland development,however,the mechanism by which it might regulate mammary gland development is unclear.The influence of LA on milk production,nutrient digestibility and the expression of proteins related to mammary gland development in dairy cows were evaluated.Forty primiparous Holstein dairy cows were divided into 4 groups in a randomized block design.Four treatments included the control(0 g/d LA per cow),low-LA(100 g/d LA per cow),medium-LA(200 g/d LA per cow),and high-LA(300 g/d LA per cow).Yields of milk,fat-corrected milk,and energy-corrected milk quadratically increased(P<0.05),and yield and content of milk fat linearly increased(P<0.05)with LA supplementation.Percentages of C12:0,C18:1 and C20:1 fatty acids in milk fat linearly increased(P<0.05),but that of C16:0 fatty acid linearly decreased(P=0.046).Supplementation of LA led to a linear and quadratical increase(P<0.05)in digestibility of dry matter,organic matter,neutral detergent fibre and acid detergent fibre,and ruminal total volatile fatty acid concentration but a linear reduction(P=0.018)in the ratio of acetate to propionate.The enzymatic activities of ruminal pectinase,xylanase,andα-amylase,and populations of total bacteria and anaerobic fungi increased linearly(P<0.05),while populations of total protozoa and methanogens decreased linearly(P<0.05)with increased LA addition.Following LA addition,blood glucose,triglyceride,estradiol,prolactin,and insulin-like growth factor 1 concentrations increased linearly(P<0.05)and albumin and total protein concentrations increased quadratically(P<0.05).Moreover,addition of 200 g/d LA promoted(P<0.05)the expression of protein involved in mammary gland development and fatty acids synthesis.These results suggested that LA addition enhanced milk production and fatty acids synthesis by stimulating nutrient digestion,the expression of proteins associated with milk fat synthesis and mammary gland development.

The Effect of Administration of Rutin on Plasma Levels of Estrogen, Prolactin, Growth Hormone and Gene Expression of Their Receptors in Mammary Glands in Ovariectomized Rats

The development of mammary glands, endocrine hormone concentrations and the gene expression of related receptors were measured in ovariectomized virgin rats after adminstration of an estrogen-like plant extract, rutin. Thirty-two ovariectomized virgin Wistar rats were randomly assigned to 4 treatments with 8 animals each： gastric infusion of 2 mL normal saline per unovariectomized rat per day （Sham）, gastric infusion of 2 mL normal saline per ovariectomized rat per day （Ova）, gastric infusion of 60 mg rutin kg-1 body weight （BW） per ovariectomized rat per day （Ova＋Rut）, or intramuscular injection of 60 ug estradiol kg-1 BW per ovariectomized rat weekly （Ova＋Est）. Samples of blood and mammary glands were harvested to determine the levels of estrogen （E2）, prolactin （PRL） and growth hormone （GH）, and the gene expression of estrogen receptors （ER）, prolactin receptors （PRLR） and growth hormone receptors （GHR） with radioimmunoassy （RIA） and RT-PCR technology, respectively. The E2 concentration in plasma and gland tissues from the rats of Ovx＋Rut or Ovx＋Est was higher than that of Ovx （P〈0.05）, but the plasma E2 concentration from the rats of Ovx＋Rut was lower than that of Sham （P〈0.05）. The order of the PRL concentration in plasma and gland tissues was Ovx〈Ovx＋Rut〈Ovx＋Est 〈Sham, and the difference in each treatment （P〈0.05）. The plasma GH concentration was lower in Ovx than in Ovx＋Rut or Ovx＋Est, and lower in Ovx＋Rut than in Sham （P〈0.05）. The GH concentration in gland tissues was lower in Ovx than in Ovx＋Rut or Ovx＋Est （P〈0.05）, and lower in Ovx＋Rut than in Sham （P〈0.05）. The gene expression of ER in gland tissues was increased in an order as Ovx〈Ovx＋Rut〈Ovx＋Est〈Sham （P〈0.05）, and PRLR, GHR showed the same trend. In conclusion, adminstration of rutin increased the E2 concentration in plasma and mammary glands, promoted pituitary PRL and GH release, up-regulated the gene expression of ER, PRLR and GHR, and stimulated mammary development in ovariectomized virgin rats.

Dietary supplementation of sodium butyrate enhances lactation performance by promoting nutrient digestion and mammary gland development in dairy cows

This experiment was to evaluate the influence of sodium butyrate(SB)addition on milk production,ruminal fermentation,nutrient digestion,and the development and metabolism regulation of the mammary gland in dairy cows.Forty Holstein dairy cows averaging 710±18.5 kg body weight,72.8±3.66 d in milk(DIM),and 41.4±1.42 kg/d milk production were divided into four treatments blocked by DIM and milk production.Treatments were control group,low SB,medium SB,and high SB with 0,100,200 and 300 g/d of SB addition per cow,respectively.The study lasted for 105 d.Production of milk,milk protein and lactose quadratically increased(P<0.05),while fat-corrected milk,energycorrected milk and milk fat yields linearly increased(P<0.05)with increasing SB addition.The digestibility of dietary dry matter,organic matter,and crude protein linearly increased(P<0.05),whereas the digestibility of ether extract,neutral detergent fibre,and acid detergent fibre quadratically increased(P<0.05).Ruminal pH quadratically decreased(P=0.04),while total volatile fatty acids(VFA)quadratically increased(P=0.03)with increasing SB addition.The acetic acid to propionic acid ratio increased(P=0.03)linearly due to the unaltered acetic acid molar percentage and a linear decrease in propionic acid molar percentage.Ruminal enzymatic activity of carboxymethyl-cellulase and a-amylase,populations of total bacteria,total anaerobic fungi,total protozoa,Ruminococcus albus,R.flavefaciens,Butyrivibrio fibrisolvens,Fibrobacter succinogenes,and Ruminobacter amylophilus linearly increased(P<0.05).Blood glucose,urea nitrogen,and non-esterified fatty acids linearly decreased(P<0.05),while total protein concentration linearly increased(P=0.04).Moreover,the addition of SB at 200 g/d promoted(P<0.05)mRNA and protein expression of PPARγ,SREBF1,ACACA,FASN,SCD,CCNA2,CCND1,PCNA,Bcl-2,GPR41,and the ratios of p-Akt/Akt and p-mTOR/mTOR,but decreased(P<0.05)mRNA and protein expressions of Bax,caspase-3,and caspase-9.The results suggest that milk production and milk fat synthesis increased with SB addition by stimulating rumen fermentation,nutrient digestion,gene and protein expressions concerned with milk fat synthesis and mammary gland development.

Maternal Bisphenol B Exposure and Mammary Gland Development of Offspring:A Time-Series Analysis

Breast cancer incidence has increased and become the world’s most prevalent cancer,which is related to abnormal development of mammary glands and thought to be influenced by environment endocrine disruptors such as bisphenol A(BPA).However,whether its substitution,bisphenol B(BPB),has similar effects remains a concern.In the present study,a maternal exposure model of ICR mice combined time-series RNA-seq analysis was established to explore the underlying correlation among maternal BPB exposure(300μg/kg body weight),mammary gland development,and long-term breast health in offspring.The results showed that BPB exposure disrupted hormonal homeostasis of the female offspring but did not affect the branch development of mammary glands in a time-dependent manner.However,at postnatal day 90(PND90),BPB exposure resulted in duct dilatation,lobular hyperplasia,and inflammatory cell infiltration and increased the number of hormone receptor-expressing(HR+)luminal cells in offspring.Further,the differentially expressed genes in time-series analysis of RNA-seq for mammary glands of the female offspring were enriched in the morphogenesis of branching structures,branching epithelium,and branching morphogenesis of epithelial tubes,which are always considered gland development.Interestingly,the results of RNA-seq also suggested that progesterone receptor(Pgr)mRNA expression in the BPB group was elevated at PND90,and breast cancer related genes such as GATA binding protein 3(Gata3)and epidermal growth factor receptor(Egfr)were also altered.These findings suggested that maternal BPB exposure did not accelerate mammary gland development or lead to obvious morphological anomalies of offspring,but it induced pathological changes and altered cancer related gene expression in adult offspring breast.