Bisphenol A and benzophenone-3 exposure alters milk protein expression and its transcriptional regulation during functional differentiation of the mammary gland in vitro

Bisphenol A (BPA) is one of the ubiquitous environmental endocrine disruptors (EEDs). Previous studies have shown that the reproduction toxicity of BPA could cause severe effects on the mammal oocytes and disturb the quality of mature oocytes. However, the toxic effects of BPA on the organelles of mouse oocytes have not been reported. In this study, to investigate whether BPA can be toxic to the organelles, we used different concentrations of BPA (50, 100, and 200 μM) to culture mouse oocytes in vitro. The results showed that 100 μM BPA exposure could significantly decrease the developmental capacity of oocytes. Then, we used the immunofluorescence staining, confocal microscopy, and western blotting to investigate the toxic effects of BPA on the organelles. The results revealed that mitochondrial dysfunction is manifested by abnormal distribution and decreased mitochondrial membrane potential. Moreover, the endoplasmic reticulum (ER) is abnormally distributed which is accompanied by ER stress showing increased expression of GRP78. For the Golgi apparatus, BPA-exposed dose not disorder the Golgi apparatus distribution but caused abnormal structure of Golgi apparatus, which is manifested by the decrease of GM130 protein expression. Moreover, we also found that BPA-exposed led to the damage of lysosome, which were shown by the increase of LAMP2 protein expression. Collectively, our findings demonstrated that the exposure of BPA could damage the normal function of the organelles, which may explain the reduced maturation quality of oocytes.

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Dissociation of cytological and functional differential in virgin mouse mammary gland during inhibition of DNA synthesis

Journal of Cell Science ◽

10.1242/jcs.53.1.97 ◽

1982 ◽

Vol 53 (1) ◽

pp. 97-114 ◽

Cited By ~ 1

Author(s):

B.K. Vonderhaar ◽

G.H. Smith

Keyword(s):

Mammary Gland ◽

Epithelial Cells ◽

Dna Synthesis ◽

Milk Protein ◽

Light And Electron Microscopy ◽

Mouse Mammary Gland ◽

Enhanced Production ◽

Virgin Mouse ◽

In Cells

Epithelial cells in mammary gland explants from mice assume a secretory appearance and synthesize the milk proteins, casein and alpha-lactalbumin, when cultured in the presence of insulin, hydrocortisone and prolactin. In cells from the glands of mature virgin animals such syntheses are known to require DNA synthesis. Addition of cytosine-beta-D-arabinofuranoside to the explant cultures suppresses both hormonally induced DNA synthesis and enhanced production of milk protein. To determine the level at which this block in terminal differentiation occurs, epithelial cell pellets were prepared from virgin mouse mammary gland explants cultured with various combination of insulin, hydrocortisone and prolactin, and subsequently examined by light and electron microscopy. We observed that the epithelial cells cultured in the presence of all three hormones developed fully, cytologically and ultrastructurally, even in the absence of DNA synthesis in vitro. Likewise, these cells were able to incorporate [3H]uridine into RNA efficiently and to incorporate amino acids into acid-precipitable polypeptides at levels equivalent to the untreated controls. However, immunoprecipitation of newly synthesized casein peptides showed that no new synthesis of casein occurred in cells prevented from synthesizing DNA. These data show uncoupling of cytological development and synthesis of milk protein in mammary explants from mature virgin mice inhibited from synthesizing DNA.

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Hormone-dependent development of milk protein synthesis in mammary gland in vitro

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/0304-4165(66)90245-5 ◽

1966 ◽

Vol 130 (2) ◽

pp. 493-501 ◽

Cited By ~ 69

Author(s):

Dean H. Lockwood ◽

Roger W. Turkington ◽

Yale J. Topper

Keyword(s):

Protein Synthesis ◽

Mammary Gland ◽

Milk Protein ◽

Dependent Development

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Arginine Supply Impacts the Expression of Candidate microRNA Controlling Milk Casein Yield in Bovine Mammary Tissue

Animals ◽

10.3390/ani10050797 ◽

2020 ◽

Vol 10 (5) ◽

pp. 797 ◽

Cited By ~ 1

Author(s):

Xin Zhang ◽

Yifan Wang ◽

Mengzhi Wang ◽

Gang Zhou ◽

Lianmin Chen ◽

...

Keyword(s):

Mammary Gland ◽

Epithelial Cells ◽

Milk Protein ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

In Vivo Study ◽

Mammary Tissue ◽

Casein Synthesis

Arginine, a semi-essential functional amino acid, has been found to promote the synthesis of casein in mammary epithelial cells to some extent. Data from mouse indicated that microRNA (miRNA) are important in regulating the development of mammary gland and milk protein synthesis. Whether there are potential links among arginine, miRNA and casein synthesis in bovine mammary gland is uncertain. The objective of the present work was to detect the effects of arginine supplementation on the expression of miRNA associated with casein synthesis in mammary tissue and mammary epithelial cells (BMEC). The first study with bovine mammary epithelial cells (BMEC) focused on screening for miRNA candidates associated with the regulation of casein production by arginine. The BMEC were cultured with three different media, containing 0, 1.6 and 3.2 mM arginine, for 24 h. The expression of candidate miRNA was evaluated. Subsequently, in an in vivo study, 6 Chinese Holstein dairy cows with similar BW (mean ± SE) (512.0 ± 19.6 kg), parity (3), BCS (4.0) and DIM (190 ± 10.3 d) were randomly assigned to three experimental groups. The experimental cows received an infusion of casein, arginine (casein plus double the concentration of arginine in casein), and alanine (casein plus alanine, i.e., iso-nitrogenous to the arginine group) in a replicated 3 × 3 Latin square design with 22 d for each period (7 d for infusion and 15 d for washout). Mammary gland biopsies were obtained from each cow at the end of each infusion period. Results of the in vitro study showed differences between experimental groups and the control group for the expression of nine miRNA: miR-743a, miR-543, miR-101a, miR-760-3p, miR-1954, miR-712, miR-574-5p, miR-468 and miR-875-3p. The in vivo study showed that arginine infusion promoted milk protein content, casein yield and the expression of CSN1S1 and CSN1S2. Furthermore, the expression of miR-743a, miR-543, miR-101a, miR-760-3p, miR-1954, and miR-712 was also greater in response to arginine injection compared with the control or alanine group. Overall, results both in vivo and in vitro revealed that arginine might partly influence casein yield by altering the expression of 6 miRNAs (miR-743a, miR-543, miR-101a, miR-760-3p, miR-1954, and miR-712).

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Bacterial Endotoxin Induces Oxidative Stress and Reduces Milk Protein Expression and Hypoxia in the Mouse Mammary Gland

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/3894309 ◽

2020 ◽

Vol 2020 ◽

pp. 1-16 ◽

Cited By ~ 1

Author(s):

Alexander Jonathan Spitzer ◽

Qing Tian ◽

Ratan K. Choudhary ◽

Feng-Qi Zhao

Keyword(s):

Oxidative Stress ◽

Mammary Gland ◽

Protein Expression ◽

Oxygen Tension ◽

Cell Apoptosis ◽

Milk Protein ◽

Staining Intensity ◽

Bacterial Endotoxin ◽

Lps Challenge ◽

Lps Treatment

The aim of this study was to investigate the mechanisms underlying the reduced milk production during mastitis. We hypothesized that bacterial endotoxin induces hypoxia, oxidative stress, and cell apoptosis while inhibiting milk gene expression in the mammary gland. To test this hypothesis, the left and right sides of the 4th pair of mouse mammary glands were alternatively injected with either lipopolysaccharide (LPS, E. coli 055: B5, 100 μL of 0.2 mg/mL) or sterile PBS through the teat meatus 3 days postpartum. At 10.5 and 22.5 h postinjection, pimonidazole HCl, a hypoxyprobe, was injected intraperitoneally. At 12 or 24 h after the LPS injection, the 4th glands were individually collected (n=8) and analyzed. LPS treatment induced mammary inflammation at both 12 and 24 h but promoted cell apoptosis only at 12 h. Consistently, H2O2 content was increased at 12 h (P<0.01), but dropped dramatically at 24 h (P<0.01) in the LPS-treated gland. Nevertheless, the total antioxidative capacity in tissue tended to be decreased by LPS at both 12 and 24 h (P=0.07 and 0.06, respectively). In agreement with these findings, LPS increased or tended to increase the mRNA expression of antioxidative genes Nqo1 at 12 h (P=0.05) and SLC7A11 at 24 h (P=0.08). In addition, LPS inhibited mammary expression of Csn2 and Lalba across time and protein expression of Csn1s1 at 24 h (P<0.05). Furthermore, hypoxyprobe staining intensity was greater in the alveoli of the PBS-treated gland than the LPS-treated gland at both 12 and 24 h, demonstrating a rise in oxygen tension by LPS treatment. In summary, our observations indicated that while intramammary LPS challenge incurs inflammation, it induces oxidative stress, increases cell apoptosis and oxygen tension, and differentially inhibits the milk protein expression in the mammary gland.

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INCORPORATION OF RAT PROLACTIN INTO RAT MILK IN VIVO AND IN VITRO

Journal of Endocrinology ◽

10.1677/joe.0.0700001 ◽

1976 ◽

Vol 70 (1) ◽

pp. 1-9 ◽

Cited By ~ 30

Author(s):

C. E. GROSVENOR ◽

N. S. WHITWORTH

Keyword(s):

Mammary Gland ◽

Trichloroacetic Acid ◽

Milk Protein ◽

Culture Media ◽

Direct Relation ◽

Excretory Organ ◽

Rat Mammary Gland ◽

Pituitary Prolactin

SUMMARY Rat prolactin (11 i.u./mg) was continuously infused into the circulation of urethaneanaesthetized lactating rats for 35 min at doses of either 200 or 472 ng/min. The immunoreactive prolactin in both milk and plasma rose quickly during the first 20–25 min of infusion, then stabilized at similar levels over baseline (68 and 98 ng/ml for milk and plasma, respectively, with the 200 ng/min dose and 250 and 230 ng/ml, respectively, with the 472 ng/min dose). The concentration of prolactin in plasma fell after the infusion was stopped, whereas that in the milk either did not fall at all or fell slightly to a new stabilized level. There was a rapid and extensive loss in the immunoreactivity of prolactin added to milk when rat milk was incubated in vitro (37 °C for 1–120 min) with either 600 ng/ml of extracted pituitary prolactin (NIAMDD RP-1) or unit equivalent amounts of prolactin obtained from pituitary culture media (secreted prolactin, supplied by C. S. Nicoll). Significantly greater amounts of added RP-1 prolactin were lost when it was incubated with milk obtained after 4 h than after 18 h of non-suckling. There was, however, no drop in endogenous immunoreactive milk prolactin levels (350–400 ng/ml) when rat milk was incubated with saline for 30 min. This suggests that milk prolactin obtained as a result of plasma transfer is different chemically from the milk prolactin resulting from the addition of either RP-1 or secreted prolactin to milk in vitro. Approximately 90% of 131I-labelled rat prolactin appeared in the trichloroacetic acid precipitable fraction after incubation (37 °C for 120 min) with milk obtained after 4 h of non-suckling in either the presence or absence of thiouracil (added to prevent binding of 131I or 131I-labelled fragments to milk protein). The recovery was slightly less when 131I-labelled prolactin was incubated with milk obtained after 18 h of non-suckling. These data suggest that prolactin is quickly transferred from plasma into milk in direct relation to the plasma concentration. Once there, much of it appears to be retained by the milk perhaps chemically or physically bound; there is little, if any, degradation of the hormone. We conclude that the lactating rat mammary gland may function normally as an excretory organ for prolactin.

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