Proteins In Mammary Gland Research Articles

Hydroxy-selenomethionine helps cows to overcome heat stress by enhancing antioxidant capacity and alleviating blood-milk barrier damage

Heat stress can lead to decreased feed intake, apoptosis of mammary epithelial cells, and decreased milk yield and quality. Selenium is an important element in the composition of at least 25 selenoproteins. Hydroxy-selenomethionine (HMSeBA; Andy Su Life Science Products Co., Ltd., Shanghai, China) is a novel organic selenium that has been shown to have a better deposition effect. However, whether HMSeBA alleviates damage to the mammary gland blood-milk barrier caused by heat stress and how this affects the performance of dairy cows remain largely unexplored. Therefore, 32 healthy Holstein cows with similar gestation days (150.41 ± 20.07 d), milk yield (36.15 ± 3.02 kg) and parity (3.25 ± 0.51) were selected and randomly divided into two total mixed rations with different selenium (Se) sources: sodium selenite (SSe) and HMSeBA. This study evaluated the outcomes of HMSeBA on antioxidant capacity, immunity, and blood-milk barrier damage in dairy cows during heat stress by collecting the samples of blood, rumen fluid and mammary gland biopsy. The experiment was conducted over 35 d, including a 5-day pre-feeding period and a 30-day experimental period. The temperature and humidity index (THI) were all above 80 throughout the experiment period. The results showed that HMSeBA decreased the respiratory rate (P < 0.001) and the content of inflammatory cytokines in the serum and increased the content of immune factors and antioxidant capacity (P < 0.05). In addition, HMSeBA reduced the expression of inflammatory cytokines and heat shock proteins in mammary gland (P < 0.05). Hematoxylin-eosin-stained pathological sections showed massive thickening of acinar walls and severe destruction of glandular structures in the SSe group, but the structure of the acinar mammary gland in the HMSeBA group was intact. Furthermore, HMSeBA promoted the expression of the phosphatidylinositol 3-kinase (PI3K, P < 0.001)/protein kinase B (AKT, P = 0.011)/mammalian target of rapamycin (mTOR, P = 0.008) pathway and improved the expression of zonula occludens-1 (ZO-1, P = 0.014) and occluding (OCLN, P = 0.012) in the mammary gland, suggesting less damage caused by heat stress to the blood-milk barrier. Our results demonstrated that HMSeBA can improve the antioxidant capacity and immunity of dairy cows and the expression of tight junction proteins in mammary gland to help alleviate the blood-milk barrier damage by heat stress, which could reduce the damage of heat stress on milk yield.

Subacute tributyltin exposure alters the development and morphology of mammary glands in association with CYP19A1 expression in female rats

Tributyltin (TBT) is an endocrine-disrupting chemical (EDC) related to reproductive dysfunctions. However, few studies have investigated the effects of TBT exposure on mammary gland development. Thus, we assessed whether subacute TBT exposure causes irregularities in mammary gland development. We administered TBT (100 and 1,000 ng/kg/day for 30 days) to female rats from postnatal day (PND) 25 to PND 55, and mammary gland development, morphology, inflammation, collagen deposition, and protein expression were evaluated. Abnormal mammary gland development was observed in both TBT groups. Specifically, TBT exposure reduced the number of terminal end buds (TEBs), type 1 (AB1) alveolar buds, and type 2 (AB2) alveolar buds. An increase in the lobule and differentiation (DF) 2 score was found in the mammary glands of TBT rats. TBT exposure increased mammary gland blood vessels, mast cell numbers, and collagen deposition. Additionally, both TBT rats exhibited intraductal hyperplasia and TEB-like structures. An increase in estrogen receptor alpha (ERα), progesterone receptor (PR), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) - positive cells was observed in the mammary glands of TBT rats. A strong negative correlation was observed between CYP19A1- positive cells and TEB number. In addition, CYP19A1 - positive cells were positively correlated with mammary gland TEB-like structure, ductal hyperplasia, inflammation, and collagen deposition. Thus, these data suggest that TBT exposure impairs mammary gland development through the modulation of CYP19A1 signaling pathways in female rats.

Amplifying local serotonin signaling prior to dry-off hastens mammary gland involution and redevelopment in dairy cows

The monoamine serotonin (5-hydroxytryptamine, 5-HT) has been reported to inhibit milk protein gene expression and increase mammary epithelial cell (MEC) tight junction permeability after milk stasis. We hypothesized that increasing serotonin synthesis and signaling within the mammary epithelium before milk stasis would increase systemic and local involution markers, and downregulate the expression of milk protein and tight junction during involution, leading to more efficient tissue growth during the redevelopment phase. Herein, we examined the outcomes of increasing local mammary 5-HT synthesis before milk stasis on involution biomarkers, mammary gland microstructure, and gene and protein expression during the dry period. Multiparous Holstein cows were administered intramammary infusions (via the teat canal) of sterile water (CON, 4 mL/teat, n = 7) or 5-hydroxy-l-tryptophan (5-HTP, serotonin precursor, 20 mg/teat, n = 7) once daily for 5 d before dry-off (d 0). Blood, milk, and mammary secretions were collected and analyzed for components and metabolites. Mammary secretions were collected 12 h after the last milking and on d 1 to 4 during the dry period at 1200 h. Mammary gland biopsies were performed on d 4 (i.e., involution phase) and d 36 (i.e., redevelopment phase) of the dry period for histological and molecular evaluation. Milk protein and tight junction gene expression was quantified via real-time PCR. Hematoxylin and eosin staining, immunohistochemistry (Ki67), and immunofluorescence (serotonin, cleaved caspase 3) were performed to visualize tissue microstructure and to quantify serotonin intensity and cell turnover. Data were analyzed in SAS (SAS Institute Inc.) using 2-way ANOVA. After d 0, mammary secretions of 5-HTP cows had increased concentrations of 5-HT, lactoferrin, and bovine serum albumin. On d 1, 5-HTP cows had greater α-lactalbumin concentrations in plasma relative to CON. Serotonin intensity was increased in the mammary tissue of 5-HTP cows on d 4, relative to CON. On d 4, milk protein and tight junction gene expression was downregulated, MEC number was reduced, and cleaved caspase 3 protein was greater in mammary tissue of 5-HTP cows, relative to CON. On d 36, milk protein genes were upregulated, and the lumen:outer alveolar area and Ki67-positive cells were increased in the mammary tissue of 5-HTP cows, relative to CON. Amplifying serotonin signaling in the mammary epithelium before milk stasis at dry-off achieves greater apoptosis, leading to a reduction in MEC, allowing for greater cell proliferation, which results in more MEC during the redevelopment phase preceding the onset of lactation.

Sodium acetate regulates milk fat synthesis through the activation of GPR41/GPR43 signaling pathway.

Fat is a critical component in milk, which provided energy for the early growth and development of mammals. Milk fat is positively related to the concentration of acetate in the blood, while the underlying mechanism is still unclear. This study is to investigate the effects of sodium acetate (NaAc) on milk fat synthesis in the mammary gland, and explored the underlying mechanism. In vitro experiments were carried out in mouse mammary epithelial cell line (HC11) cells cultured with NaAc to explore the potential pathway of NaAc on milk fat synthesis. Furthermore, 24 pregnant mice (from d 18.5 of gestation to d 7 of lactation, exposed to 200 mM NaAc drinking water) were used as an in vivo model to verify the results. In this study, we found that NaAc promoted milk fat synthesis and the expression of related genes and proteins in HC11 mammary epithelial cells with the activation of GPCR and mTORC1 signaling pathways (p < 0.05). Pretreatment with the mTORC1 inhibitors and G protein inhibitors attenuated the NaAc-induced milk fat synthesis in HC11 mammary epithelial cells (p < 0.05). Importantly, the effect of NaAc on milk synthesis was attenuated in GPR41 and GPR43 knockdown HC11 mammary epithelial cells (p < 0.05). This evidence indicates that NaAc might regulate milk fat synthesis through the GPR41/GPR43-mTORC1 pathway. Consistently, in in vivo experiment, dietary supplementation with NaAc significantly increased milk fat content and fat synthesis-related proteins in mice mammary glands with the activation of mTORC1 and GPCR signaling pathways at peak lactation (p < 0.05). The addition of NaAc promoted the increase of milk fat synthesis in HC11 mammary epithelial cells and mice mammary glands at peak lactation. Mechanistically, NaAc activates GPR41 and GPR43 receptors, leading to the activation of the mTORC1 signaling pathway to promote the synthesis of milk fat.Graphical abstract.

An Integrated Analysis of Lactation-Related miRNA and mRNA Expression Profiles in Donkey Mammary Glands.

Donkey milk is consumed by humans for its nutritional and therapeutic properties. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) have been implicated in the regulation of milk component synthesis and mammary gland development. However, the regulatory profile of the miRNAs and mRNAs involved in lactation in donkeys is unclear. We performed mRNA-seq and miRNA-seq and constructed coexpression regulatory networks for the mammary glands during the lactating and nonlactating period of jennies. We identified 3144 differentially expressed (DE) mRNAs (987 upregulated mRNAs and 2157 downregulated mRNAs) and 293 DE miRNAs (231 upregulated miRNAs and 62 downregulated miRNAs) in the lactating group compared to the nonlactating group. The DE miRNA target mRNA were significantly associated with pathways related to RNA polymerase, glycosphingolipid biosynthesis, mRNA surveillance, ribosome biogenesis in eukaryotes, glycerophospholipid metabolism, Ras signaling, and the fly hippo signaling pathway. The mRNA–miRNA coregulation analysis showed that novel-m0032-3p, miR-195, miR-26-5p, miR-23-3p, miR-674-3p, and miR-874-3p are key miRNAs that target mRNAs involved in immunity and milk lipid, protein, and vitamin metabolism in the jenny mammary gland. Our results improve the current knowledge of the molecular mechanisms regulating bioactive milk component metabolism in the mammary glands and could be used to improve milk production in donkeys.

Increased supply of methionine during a heat-stress challenge in lactating holstein cows alters mammary tissue mTOR signaling and its response to lipopolysaccharide.

The first objective was to investigate the effects of feeding rumen-protected methionine (RPM) during a heat stress (HS) challenge on abundance and phosphorylation of mechanistic target of rapamycin (mTOR)-related signaling proteins in mammary gland. The second objective was to investigate how HS and RPM may modulate the response of mammary gland explants to an inflammatory challenge using lipopolysaccharide (LPS). Thirty-two multiparous, lactating Holstein cows (184 ± 59 DIM) were randomly assigned to 1 of 2 environmental treatment groups, and 1 of 2 dietary treatments [TMR with RPM (Smartamine M; Adisseo Inc.; 0.105% DM as top dress) or TMR without RPM (CON)] in a crossover design. There were two periods with two phases per period. In phase 1 (9 d), all cows were in thermoneutral conditions (TN) and fed ad libitum. During phase 2 (9 d), group 1 (n = 16) cows were exposed to HS using electric heat blankets, whereas group 2 cows (n = 16) remained in TN but were pair-fed to HS counterparts to control for DMI decreases associated with HS. After a washout period (14 d), the study was repeated (period 2). Environmental treatments were inverted in period 2 (sequence), whereas dietary treatments remained the same. Mammary tissue was harvested via biopsy at the end of both periods. Tissue was used for protein abundance analysis and also for incubation with 0 or 3 μg/mL of LPS for 2 h and subsequently used for mRNA abundance. Data were analyzed using PROC MIXED in SAS. Analysis of protein abundance data included the effects of diet, environment and their interaction, and period and sequence to account for the crossover design. The explant data model also included the effect of LPS and its interaction with environment and diet. Abundance of phosphorylated mTOR and ratio of phosphorylated eukaryotic translation elongation factor 2 (p-EEF2) to total EEF2 in non-challenged tissue was greater with RPM supplementation (P = 0.04 for both) and in both cases tended to be greater with HS (P = 0.08 for both). Regardless of RPM supplementation, incubation with LPS upregulated mRNA abundance of IL8, IL6, IL1B, CXCL2, TNF, NFKB1, and TLR2 (P < 0.05). An environment × LPS interaction was observed for NFKB1 (P = 0.03); abundance was greater in LPS-treated explants from non-HS compared with HS cows. Abundance of CXCL2, NFKB1, NOS2, NOS1, and SOD2 was lower with HS (P < 0.05). Although LPS did not alter mRNA abundance of the antioxidant transcription factor NFE2L2 (P = 0.59), explants from HS cows had lower abundance of NFE2L2 (P < 0.001) and CUL3 (P = 0.04). Overall, RPM supplementation may alter mTOR activation in mammary tissue. Additionally, although HS reduced explant immune and antioxidant responses, RPM did not attenuate the inflammatory response induced by LPS in vitro.

Mammary Development in Gilts at One Week Postnatal Is Related to Plasma Lysine Concentration at 24 h after Birth, but Not Colostrum Dose.

Simple SummaryA relationship exists between a female’s early nutritional environment and her ability to produce milk when she lactates as an adult. Colostrum is the first milk available to neonates after birth. We hypothesized that differing levels of colostrum stimulate differences in very early mammary development. Despite differences in weight at 24 h and 7 days, mammary morphological development and DNA content was not found to be different between gilts fed a high versus low dose of colostrum. The rate of mammary gland protein and DNA synthesis over the first week was not different between the groups. Circulating levels of amino acids were determined after 24 h of colostrum feeding, and levels of circulating lysine were found to be related to average daily gain and mammary DNA synthetic rate. Moreover, the level of lysine was related to a lower ratio of DNA to protein synthesis, suggesting that higher lysine favored cell division versus differentiation (by leaving the cell cycle). Further studies are needed in this area.Perinatal nutrition affects future milk production. The number of mammary epithelial cells affect milk production capacity. Therefore, it was hypothesized that the level of colostrum intake affects the proliferation rate and the total number of mammary epithelial cells in the gland. The ratio of newly synthesized protein to newly synthesized DNA reflects the relative amount of cellular differentiation to cell division. The study objective was to determine the relationship between the level of colostrum intake and 24 h-level of circulating amino acid, glucose and insulin with mammary parenchyma histological features, cell division and protein synthesis over the first week postnatal. One of two standardized doses of a homogenate colostrum sample, 10% (n = 8) and 20% (n = 8) of birth bodyweight, was fed to gilts over the first 24 h postnatal. Gilts were administered deuterium oxide immediately after birth and daily to label newly synthesized DNA and proteins. Gilts were euthanized on postnatal day seven, and DNA and protein were isolated from mammary parenchyma. DNA and protein fractional synthesis (f) and fractional synthetic rate (FSR) were calculated using mass isotopomer distribution analysis. The ratio of protein f and FSR to DNA f and FSR were calculated and used to indicate the relative amounts of differentiation to cell division. Mammary morphological development was also analyzed by measuring the parenchymal epithelial area and the stromal and epithelial proliferation index on postnatal day seven. Colostrum dose was not related to any of the variables used to evaluate mammary development. However, plasma lysine levels at 24 h postnatal were positively related to average daily gain (ADG; r = 0.54, p = 0.05), DNA f (r = 0.57; p = 0.03) and DNA FSR (r = 0.57; p = 0.03) in mammary parenchyma. Plasma lysine was inversely related to the ratio of protein to DNA f and FSR (r = −0.56; p = 0.04). ADG was related to the parenchymal epithelial area and DNA and protein f and FSR (p < 0.05). These relationships support the idea that the nutritional environment affects early mammary development and that higher lysine levels in the perinatal period favored a greater degree of cell division versus differentiation in mammary of neonatal pigs and thus, warrant further investigations.

Taraxacum mongolicum protects against Staphylococcus aureus-infected mastitis by exerting anti-inflammatory role via TLR2-NF-κB/MAPKs pathways in mice

Ethnopharmacological relevanceAs a traditional Chinese medicine, Taraxacum mongolicum has been widely used for the prevention and treatment of a variety of inflammatory and infectious diseases, and also clinically used as a remedy for mastitis. However, the scientific rationale and mechanism behind its use on mastitis in vivo are still unclear.Aim of the study: This study aimed to investigate the protective effect and potential mechanism of Taraxacum mongolicum Hand.-Mazz. (T. mongolicum) on mastitis infected by Staphylococcus aureus (S. aureus). Materials and methodsFemale ICR mice were given intragastrically 2.5, 5 and 10 g/kg of T. mongolicum extract twice per day for 6 consecutive days, and infected with S. aureus via teat canal to induce mastitis. Pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) levels were determined by ELISA. Myeloperoxidase (MPO) activity and distribution were measured by reagent kit and immunohistochemistry. Histopathological changes of mammary gland tissues were observed by H&E staining. Toll-like receptor 2 (TLR2) expression, phosphorylations of related proteins in nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways were detected by western blot. ResultsT. mongolicum decreased TNF-α, IL-6 and IL-1β levels, and reduced MPO activity and distribution in sera and mammary glands with S. aureus-infected mastitis. In addition, T. mongolicum effectively attenuated histopathological damages and cell necrosis of mammary gland tissues infected by S. aureus. Moreover, T. mongolicum inhibited the expression of TLR2, and the phosphorylations of inhibitor κBα (IκBα), p65, p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) proteins in mammary glands with S. aureus-infected mastitis. ConclusionsThis study suggests that T. mongolicum protects against S. aureus-infected mastitis by exerting anti-inflammatory role, which is attributed to the inhibition of TLR2-NF-κB/MAPKs pathways.

Лагодюк Петро Захарович — доктор біологічних наук, професор, академік НААН, Заслужений діяч науки і техніки України (1924–1994)

Petro Lahodyuk is the Doctor of Biological Sciences, professor, Academician of NAAS, Honourable Man of Science and Technology of Ukraine. Academician Lahodyuk was a leading researcher of animal lactation physiology. He contributed greatly into research on fraction composition and antigene properties of soluble proteins in mammary gland of open heifers, heifers and lactating cows, open and pregnant cows, compared their immune and chemical properties with milk and blood serum proteins, researched amino acidic and peptoid content of albumins in mammary gland tissues and blood serum of open heifers, heifers and cows, established the role of alveolar epithelium, excretory ducts and milk ducts in creating milk serum proteins, studied the role of a number of hormones in milk formation processes regulation, in particular regulation of milk and blood serum protein biosynthesis.

UFM1-Specific Ligase 1 Ligating Enzyme 1 Mediates Milk Protein and Fat Synthesis-Related Gene Expression via the JNK Signaling Pathway in Mouse Mammary Epithelial Cells.

Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1) has been characterized as a ubiquitin-like (Ubl) protein that affects a range of cellular processes across various pathways. In this study, mouse mammary epithelial cells (HC11 cell line) and UFL1 knockout (KO) mice were used to establish UFL1 knockdown models to explore the influence of UFL1 on milk protein and fat synthesis in the mouse mammary gland and the underlying mechanisms. This is the first study to show UFL1 localization in mouse mammary epithelial cells. UFL1 depletion by transfected UFL1 siRNA (siUFL1) caused aggravated apoptosis. In addition, UFL1 depletion suppressed milk protein synthesis-related protein level in vivo and in vitro. Conversely, ACACA and FASN expressions increased in UFL1-deficient mice. Moreover, UFL1 depletion increased triglyceride synthesis levels and inhibited the p-JNK expression. Importantly, the expression of proteins related to milk protein synthesis was decreased in JNK- and UFL1-deficient cells, whereas proteins related to milk fat synthesis showed the opposite trend, indicating that UFL1 affects milk protein and fat synthesis via the suppression of JNK activation. Overall, our findings indicate that UFL1 plays a key role in mammary milk and fat synthesis via JNK activation.

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

Simple SummaryIt has been reported that arginine plays an important role in lactation, including promoting mammary gland development, increasing yields of milk and casein. Recent studies revealed that microRNA could be involved in regulating expression of functional genes related to mammary development. Thus, exploring the role of arginine on the regulation of miRNA related to bovine mammary development and milk production in dairy cows is of importance. The present work revealed that arginine injection increased casein yield by altering the expression of selected microRNA associated with mammary development.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).

Valine supplementation during late pregnancy in gilts increases colostral protein synthesis through stimulating mTOR signaling pathway in mammary cells

Mammary gland development during late pregnancy in sows is a major factor affecting the composition of colostrum and milk and the pre-weaning growth of piglets, while valine is essential for protein and nitrogen metabolism in mammary gland of sow. However, the effects of valine and its underlying mechanism on mammary gland development during late pregnancy are still unclear. Here, we hypothesized that dosage of dietary valine during late pregnancy will affect protein synthesis of colostrum in gilts. The results showed that supplementation of valine during late pregnancy significantly increased content of protein (P < 0.01), fat (P = 0.02) and solids-non-fat (P = 0.04) in colostrum. Our in vitro study also confirmed that valine supplementation increased protein synthesis and cell proliferation in porcine mammary epithelial cells (PMEC). Furthermore, these changes were associated with elevated phosphorylation levels of mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase (S6) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1) in valine-supplemented cells, which could be effectively blocked by the antagonists of mTOR. These findings indicated that valine enhanced mammary gland development and protein synthesis in colostrum via the mTOR signaling pathway. These results, using an in vivo and in vitro model, helped to understand the beneficial effects of dietary valine supplementation on gilts.

Effect of Acupuncture Combined with Gavage of Rule Granule on Serum Prolactin, Estradiol and Progestone Contents and Their Receptor Expression in Breast in Mammary Gland Hyperplasia Rats

To observe the effect of acupuncture combined with medication (Rule Granule) on serum prolactin (PRL)，estradiol (E2) and progestone (P) contents and expressions of prolactin receptor (PRLR), estrogen receptor (ER) and progestrone receptor (PR) proteins in mammary gland (MG) tissues of MG hyperplasia (MGH) rats, so as to explore its mechanisms underlying improvement of MGH. A total of 55 female SD rats were randomized into blank control group (n＝10), model group (n＝10), acupuncture group (n＝11), medication group (n＝10), and acupuncture plus medication group (n＝9). The MGH model was established by muscular injection of E2 benzoate(0.5 mg/kg, once daily for 20 days), followed by injection of P (5 mg/kg) into the lateral muscle of the hind-limb, once daily for 5 days, and the rats of the control group were treated by muscular injection of normal saline at the same site and in the same procedures. Acupoint group A composed of bilateral Tianzhong (SI 11), Ganshu (BL 18) and Zusanli (ST 36), and group B composed of Tanzhong (CV 17), and bilateral Wuyi (ST 15) and Hegu (LI 4) were alternatively punctured with filiform needles and stimulated swiftly by twirling the needle in each acupoint for 20－30 sec, once daily for 30 days. Rats of the medication group and acupuncture ＋medication group were treated by gavage of Rule Granule fluid containing Baishao (Paeonia Iactiflora Pall), Danggui (Angelica sinensis), etc. (1.5 mL/100 g for each rat) and those of the other groups treated by gavage of distilled water (1.5 mL/100 g). The treatment was given once daily for continuous 30 d. At the end of the experiments, the rats' abdominal aorta blood was collected for assaying the contents of serum PRL, E2 and P with ELISA, and the pathological changes of breast tissue (the left 2nd pairs) were observed under microscope after sectioning and H．E. staining. The expression of PRLR, ER and PR proteins in the breast tissue was detected by immunohistochemistry. After modeling, the hyperplasia of mammary gland in the model group was obvious; the serum PRL and E2 contents and the immunoactivities of PRLR, ER and PR were significantly increased (P<0.01); and the content of serum P was considerably decreased in the model group in comparison with the blank control group (P<0.01). After the treatment, the hyperplasia of mammary gland was improved; the serum PRL and E2 contents and the expression of PRLR, ER and PR were significantly decreased (P<0.05, P<0.01); and the content of P was notably increased in the acupuncture, medication and acupuncture plus medication groups relevant to the model group (P<0.05，P<0.01). The therapeutic effects of acupuncture plus medication were significantly superior to those of both simple medication and simple acupuncture in down-regulating serum PRL and E2 contents and PRLR, ER and PR immunoactivity, as well as in up-regulating serum P content (P<0.05). No significant differences were found between the medication and acupuncture groups in the above mentioned 3 serum and 3 mammary indexes (P>0.05)．. Acupuncture, Rule Granule, and acupuncture combined with Rule Granule can improve hyperplasia of mammary gland in rats, which may be related to their effects in reducing serum PRL and E2 and breast ER, PR and PRLR expression levels, and in increasing serum P level. The therapeutic effects of acupuncture plus Rule Granule are better than those of simple acupuncture and simple Rule Granule.

Serotonin induces parathyroid hormone-related protein in goat mammary gland.

During lactation, large amounts of calcium are exported from the mammary gland into milk to ensure skeletal growth of the offspring. Recent studies revealed that serotonin (5-HT) is essential to stimulate skeletal calcium resorption for milk synthesis. Our objective was to explore the correlation between circulating 5-HT and serum calcium and parathyroid hormone-related protein (PTHrP) concentrations around parturition in dairy goats. We also investigated the effect of 5-HT on PTHrP expression in cultured primary goat mammary epithelial cells (GMEC). Blood samples of multiparous Guanzhong dairy goats were collected on day -5 to 3 postpartum for analysis of serum concentrations of calcium, 5-HT, and PTHrP. Results revealed that from day -3 to 0 postpartum serum calcium and 5-HT concentrations decreased progressively, but serum PTHrP concentration only had a sharp drop in the postpartum period sampled. Correlation analysis of circulating 5-HT and serum calcium and PTHrP concentrations on day 1 and 2 postpartum revealed that low serum 5-HT concentration was positively correlated with serum total calcium or PTHrP concentration. By knocking down tryptophan hydroxylase-1 (TPH1) or adding 5-hydroxytryptophan (5-HTP) to decrease or increase the levels of 5-HT in GMEC, we observed that 5-HTP increased PTHrP expression in a dose-dependent manner and siTPH1 decreased PTHrP protein expression. Furthermore, 5-HT increased mRNA abundance of calcium-sensing receptor (CaSR) in a dose-dependent manner and decreased the expression of plasma membrane Ca2+ ATPase-1 (PMCA1). Taken together, 5-HT seems to induce PTHrP expression in goat mammary cells during and after parturition. These findings suggest that increasing 5-HT biosynthesis could be a potential therapeutic target for prevention of hypocalcemia in dairy goats.

Expression of iron-related proteins in feline and canine mammary gland reveals unexpected accumulation of iron

Dysregulation of cellular iron homeostasis in human breast cancer is reflected by the altered expression of regulatory proteins. The expressions of iron-related proteins in the mammary glands of cats and dogs have not been assessed. We evaluated the expressions of ferritin, ferroportin, hepcidin and transferrin receptor 1 in benign and malignant mammary gland lesions in cats and dogs. Iron deposition was detected using Perls’ Prussian blue staining. We found no major differences in the expression of iron-related proteins between benign and malignant mammary gland lesions in either cats or dogs; however, these species exhibited accumulation of iron in benign lesions. Our findings provide an explanation for the absence of higher iron requirements by tumor cells in these animals. Further investigation of local iron homeostasis in cats and dogs and differences in their physiology compared to human breast cancer is required.

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland.

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 μg/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.

Litter-of-origin trait effects on gilt development1

The preweaning litter environment of gilts can affect subsequent development. In a recent experiment designed to test the effects of diet on gilt development, litter-of-origin traits including individual birth weights, immunocrits (a measure of colostrum intake), sow parity, number weaned, and individual weaning weights were collected for approximately 1,200 gilts that were progeny of approximately 300 sows. Subsequently, BW, LM area, and backfat were measured at 100 d of age and at 28-d intervals until slaughter (260 d of age). From 160 d of age to slaughter, gilts were observed daily for estrus. At slaughter, the reproductive tract and 1 mammary gland were recovered. The reproductive tract was classified as cyclic or prepubertal; the number of corpora lutea was counted. Uterine horn lengths and ovarian dimensions were measured. Uterus and ovary samples from every 10th gilt were prepared for histological evaluation of uterine gland development and follicle counts, respectively. Mammary gland tissue protein and fat were assayed. Day of the estrous cycle at slaughter was calculated using the first day of the most recent standing estrus (d 0) recorded previous to slaughter. Each gilt development trait was analyzed for association with each litter-of-origin trait, after adjusting for dietary treatment effects. Uterine length, ovarian dimensions, mammary gland protein and fat, and uterine gland development were also adjusted for day of the estrous cycle at slaughter. All litter-of-origin traits were associated ( < 0.05) with growth traits. Top-down (backward elimination) multiple regression analysis indicated that BW and LM accretion in gilts was positively associated with immunocrit ( < 0.01), birth weight ( < 0.01), preweaning growth rate ( < 0.01), and parity ( < 0.01). Backfat accretion was positively associated with preweaning growth rate ( < 0.01), number weaned ( < 0.05), and parity ( < 0.05). Age at puberty was associated with birth weight (positive; < 0.01) and preweaning growth rate (negative; < 0.01). Total uterine length was positively associated with only birth weights ( < 0.05). Mammary gland protein was negatively associated with preweaning growth ( < 0.01). Mammary gland fat was positively associated with birth weight and number of piglets weaned ( > 0.05). These results indicate that colostrum consumption, birth weights, preweaning growth rate, number weaned, and parity are associated with gilt development traits during later life.

Differential expression of genes in milk of dairy cattle during lactation

SummaryThe milk fat globule (MFG) is one of the most representative of mammary gland tissues and can be utilized to study gene expression of lactating cows during lactation. In this study, RNA‐seq technology was employed to detect differential expression of genes in MFGs at day 10 and day 70 after calving between two groups of cows with extremely high (H group) and low (L group) 305‐day milk yield, milk fat yield and milk protein yield. In total, 1232, 81, 429 and 178 significantly differentially expressed genes (false discovery rate q < 0.05) were detected between H10 and L10, H70 and L70, H10 and H70, and L10 and L70 respectively. Gene Ontology enrichment and pathway analysis revealed that these differentially expressed genes were enriched in biological processes involved in mammary gland development, protein and lipid metabolism process, signal transduction, cellular process, differentiation and immune function. Among these differentially expressed genes, 178 (H10 vs. L10), 4 (H70 vs. L70), 68 (H10 vs. H70) and 22 (L10 vs. L70) were found to be located within previously reported QTL regions for milk production traits. Based on these results, some promising candidate genes for milk production traits in dairy cattle were suggested.

Bergenin Plays an Anti-Inflammatory Role via the Modulation of MAPK and NF-κB Signaling Pathways in a Mouse Model of LPS-Induced Mastitis.

Mastitis is a major disease in humans and other animals and is characterized by mammary gland inflammation. It is a major disease of the dairy industry. Bergenin is an active constituent of the plants of genus Bergenia. Research indicates that bergenin has multiple biological activities, including anti-inflammatory and immunomodulatory properties. The objective of this study was to evaluate the protective effects and mechanism of bergenin on the mammary glands during lipopolysaccharide (LPS)-induced mastitis. In this study, mice were treated with LPS to induce mammary gland mastitis as a model for the disease. Bergenin treatment was initiated after LPS stimulation for 24h. The results indicated that bergenin attenuated inflammatory cell infiltration and decreased the concentration of NO, TNF-α, IL-1β, and IL-6, which were increased in LPS-induced mouse mastitis. Furthermore, bergenin downregulated the phosphorylation of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathway proteins in mammary glands with mastitis. In conclusion, bergenin reduced the expression of NO, TNF-α, IL-1β, and IL-6 proinflammatory cytokines by inhibiting the activation of the NF-κB and MAPKs signaling pathways, and it may represent a novel treatment strategy for mastitis.

Proteomics and pathway analysis of N-glycosylated mammary gland proteins in response to Escherichia coli mastitis in cattle

The aim of this study was to investigate the N-linked glycosylated protein profile of mammary tissue from healthy cows and cows with mastitis due to Escherichia coli, in order to understand the molecular mechanisms of the host response to mastitis. N-glycopeptides were enriched with a lectin mixture and identified through high-accuracy mass spectrometry. A total of 551 N-glycosylation sites, corresponding to 294 proteins, were identified in the mammary tissues of healthy cows; these glycoproteins were categorised into three functional groups and clustered into 11 specific pathways. A total of 511 N-glycosylation sites, corresponding to 283 glycosylated proteins, were detected in the mammary tissues of cows with E. coli mastitis. There were differences in N-glycosylation sites in 98 proteins in the mammary tissues of healthy cows and cows with mastitis due to E. coli. Most proteins with altered glycosylation were those involved in responses to stress, cell adhesion and the immune response, and were assigned to five specific pathways based on their gene ontology annotation. The results from this study show that the glycosylated protein profile in the mammary tissues of healthy and mastitic cows are different, and altered glycoproteins are associated with several pathways, including the lysosome and O-glycan biosynthesis pathways.