Calmodulin content of rat mammary tissue and isolated cells during pregnancy and lactation

The calmodulin content of heat-treated extracts of rat mammary tissue and isolated cells was measured by using stimulation of cyclic nucleotide phosphodiesterase (PDE) activity and radioimmunoassay (r.i.a.) procedures. The calmodulin content of mammary tissue increased 2.5-fold near the time of parturition, remained at the elevated level during lactation, then, after the onset of involution, decreased to values similar to those measured from mammary tissue of pregnant rats. When tissue from 15 animals in different stages of pregnancy, lactation and involution were compared, the r.i.a. gave 2.6-fold higher results than the PDE assay. To investigate further the increase in calmodulin content of mammary tissue, secretory and myoepithelial cells were enzymically dissociated from rat mammary tissue during different stages of pregnancy, lactation and involution. Protein, DNA, lactose, glucose-6-phosphate dehydrogenase and alkaline phosphatase were assayed to characterize the cell fractions. By using r.i.a., the calmodulin content per mg of protein in isolated secretory-cell fractions was high near parturition, then decreased and remained relatively constant during lactation. The amount of calmodulin expressed per mg of DNA in secretory cells did not show a marked change near parturition, suggesting a constant amount of calmodulin per cell. The calmodulin content of myoepithelial cells dissociated from mammary tissue and measured by using r.i.a. was 6-fold lower than in secretory cells and remained relatively constant during the course of lactation. The changing levels of calmodulin in rat mammary tissue during development are suggested to be related to proliferation and destruction of secretory epithelial cells, events that occur near parturition and involution respectively.

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Characterization of mammary cells from the lactating rat by electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083679 ◽

1978 ◽

Vol 36 (2) ◽

pp. 560-561

Author(s):

P. Sadhukhan ◽

J. Chakraborty ◽

M. S. Soloff ◽

M. H. Wieder ◽

D. Senitzer

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Cell Types ◽

Myoepithelial Cells ◽

Mammary Tissue ◽

Secretory Cells ◽

Isolated Cells ◽

Transmission Electron ◽

Cell Processes ◽

Phosphatase Cytochemistry

The means to identify cells isolated from the mammary gland of the lactating rat as a prerequisite for cell purification have been developed.The cells were isolated from mammary tissue with 0. 1% collagenase, and they were visualized by scanning and transmission electron microscopy and by alkaline phosphatase cytochemistry.The milk-secreting cells have surface microvilli, whereas the surface of the myoepithelial cells is smooth (Fig. 1). The two isolated epithelial cell types are readily distinguishable by transmission electron microscopy (Fig. 2). The secretory cells contain vacuoles and a relatively extensive rough endoplasmic reticulum, whereas the myoepithelial cells contain a more osmiophilic cytoplasm, contractile filaments (Fig. 3) and elongate processes. These features are consistent with the appearance of the two cell types in situ.Incubation of isolated cells with oxytocin prior to glutaraldehyde fixation resulted in the contraction of the myoepithelial cell processes (Figs. 4 & 5). This physiological response to oxytocin shows that the isolated myoepithelial cells were intact. The appearance of isolated secretory cells was unchanged by the presence of oxytocin.

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Oxytocin receptors in rat involuting mammary gland

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o83-079 ◽

1983 ◽

Vol 61 (7) ◽

pp. 631-635 ◽

Cited By ~ 18

Author(s):

Melvyn S. Soloff ◽

Michael H. Wieder

Keyword(s):

Alkaline Phosphatase ◽

Mammary Gland ◽

Fold Increase ◽

Myoepithelial Cells ◽

Oxytocin Receptor ◽

Secretory Cells ◽

Specific Marker ◽

Oxytocin Receptors ◽

Pregnancy And Lactation ◽

Receptor Number

Oxytocin-receptor concentrations in the rat mammary gland were determined by Scatchard analyses with [3H]oxytocin. There was about a 100-fold increase in the number of receptors per mammary gland between the 1st day of pregnancy and late lactation. The number of receptors then fell markedly during postweaning mammary regression, but rose again during a second pregnancy and lactation cycle. The changes in oxytocin-receptor number corresponded to changes in alkaline phosphatase activity per mammary gland. These results strongly support data suggesting that alkaline phosphatase, like oxytocin receptors, is a specific marker for mammary myoepithelial cells. Despite the fall in oxytocin-receptor number per mammary gland during postweaning regression, the concentration of receptors, expressed per milligram of protein, increased 10-fold over lactating levels on the 6th day of regression. Thereafter, receptor concentrations declined, but were still elevated about fivefold over lactating levels on the 15th day of regression. It is likely that the increased concentration of receptors was due to a decrease in the relative amount of nontarget secretory cells. The factors that regulate the concentration of oxytocin receptors on mammary myoepithelial cells are presently unknown; however, the involuting mammary system may be practical for obtaining enriched populations of oxytocin-sensitive myoepithelial cells.

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Nuclear targeting of stanniocalcin to mammary gland alveolar cells during pregnancy and lactation

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00098.2005 ◽

2005 ◽

Vol 289 (4) ◽

pp. E634-E642 ◽

Cited By ~ 12

Author(s):

Craig P. Hasilo ◽

Christopher R. McCudden ◽

J. Ryan J. Gillespie ◽

Kathi A. James ◽

Edward R. Hirvi ◽

...

Keyword(s):

Gene Expression ◽

Mammary Gland ◽

Molecular Mass ◽

Chemical Reduction ◽

Mammary Tissue ◽

Nuclear Targeting ◽

Alveolar Cells ◽

Hormone Production ◽

Pregnant Rats ◽

Pregnancy And Lactation

In most mammalian tissues, the stanniocalcin-1 gene (STC-1) produces a 50-kDa polypeptide hormone known as STC50. Within the ovaries, however, the STC-1 gene generates three higher-molecular-mass variants known as big STC. Big STC is targeted locally to corpus luteal cells to block progesterone release. During pregnancy and lactation, however, ovarian big STC production increases markedly, and the hormone is released into the serum. During lactation, this increase in hormone production is dependent on a suckling stimulus, suggesting that ovarian big STC may have regulatory effects on the lactating mammary gland. In this report, we have addressed this possibility. Our results revealed that virgin mammary tissue contained large numbers of membrane- and mitochondrial-associated STC receptors. However, as pregnancy progressed into lactation, there was a decline in receptor densities on both organelles and a corresponding rise in nuclear receptor density, most of which were on milk-producing, alveolar cells. This was accompanied by nuclear sequestration of the ligand. Sequestered STC resolved as one ∼135-kDa band in the native state and therefore had the appearance of a big STC variant. However, chemical reduction collapsed this one band into six closely spaced, lower-molecular-mass species (28–41 kDa). Mammary gland STC production also underwent a dramatic shift during pregnancy and lactation. High levels of STC gene expression were observed in mammary tissue from virgin and pregnant rats. However, gene expression then fell to nearly undetectable levels during lactation, coinciding with the rise in nuclear targeting. These findings have thus shown that the mammary glands are indeed targeted by STC, even in the virgin state. They have further shown that there are marked changes in this targeting pathway during pregnancy and lactation, accompanied by a switch in ligand source (endogenous to exogenous). They also represent the first example of nuclear targeting by STC.

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Production of parathyroid hormone-related protein by the rat mammary gland in pregnancy and lactation

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1992.263.6.e1077 ◽

1992 ◽

Vol 263 (6) ◽

pp. E1077-E1085 ◽

Cited By ~ 3

Author(s):

M. Rakopoulos ◽

S. J. Vargas ◽

M. T. Gillespie ◽

P. W. Ho ◽

H. Diefenbach-Jagger ◽

...

Keyword(s):

Mammary Gland ◽

Parathyroid Hormone ◽

Mammary Tissue ◽

Secretory Cells ◽

Related Protein ◽

Sprague Dawley ◽

Parathyroid Hormone Related Protein ◽

Pregnancy And Lactation ◽

Rat Mammary Gland ◽

In Pregnancy

Production of parathyroid hormone-related protein by the rat mammary gland in pregnancy and lactation. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E1077-E1085, 1992.--Production of parathyroid hormone-related protein (PTHrP) by the mammary gland of Sprague-Dawley rats has been examined using immunohistochemistry and in situ hybridization to detect PTHrP and PTHrP mRNA, respectively. PTHrP and PTHrP mRNA could be demonstrated in nests of epithelial cells of the developing mammary gland at day 14 of pregnancy and in the epithelial secretory cells lining the alveoli during the latter stages of pregnancy and during lactation. A specific radioimmunoassay was also used to measure the concentration of PTHrP secreted in the milk throughout lactation. The concentration of PTHrP in milk was relatively low initially but increased during the latter stages of lactation, whereas calcium concentrations remained virtually constant throughout lactation. No correlation was found between the concentrations of calcium and PTHrP in rat milk. These results show that PTHrP is present in rat milk and also in mammary tissue before parturition, and therefore it may assist in the development of the mammary gland during pregnancy.

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Production of parathyroid hormone-related protein by the rat mammary gland in pregnancy and lactation

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2006.263.6.e1077 ◽

2006 ◽

Vol 263 (6) ◽

pp. E1077-E1085 ◽

Cited By ~ 1

Author(s):

M. Rakopoulos ◽

S. J. Vargas ◽

M. T. Gillespie ◽

P. W. Ho ◽

H. Diefenbach-Jagger ◽

...

Keyword(s):

Mammary Gland ◽

Parathyroid Hormone ◽

Mammary Tissue ◽

Secretory Cells ◽

Related Protein ◽

Sprague Dawley ◽

Parathyroid Hormone Related Protein ◽

Pregnancy And Lactation ◽

Rat Mammary Gland ◽

In Pregnancy

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Progesterone and the metabolic control of the lactose biosynthetic pathway during lactogenesis in the rat

Biochemical Journal ◽

10.1042/bj1361105 ◽

1973 ◽

Vol 136 (4) ◽

pp. 1105-1116 ◽

Cited By ~ 23

Author(s):

Gillian Murphy ◽

Ari D. Ariyanayagam ◽

N. J. Kuhn

Keyword(s):

Adenine Nucleotide ◽

Equilibrium Constants ◽

Energy Charge ◽

Mass Action ◽

Mammary Tissue ◽

Protein Activity ◽

Pregnant Rats ◽

Metabolic Intermediates ◽

Lactose Synthesis ◽

Glucose 6 Phosphate Dehydrogenase

1. Lactogenesis was initiated in pregnant rats by ovariectomy, thereby causing progesterone withdrawal, after which the mammary tissue was analysed for contents of enzymes and metabolites concerned with the biosynthesis of lactose. 2. Lactose synthesis increased about 126-fold with little or no accompanying change in the contents of most metabolic intermediates or in the adenine nucleotide energy charge. 3. Comparison of mass-action ratios with equilibrium constants showed that phosphoglucomutase (EC 2.7.5.1), UDP-glucose pyrophosphorylase (EC 2.7.7.9) and UDP-glucose epimerase (EC 5.1.3.2.) catalysed reactions close to equilibrium. Nucleoside diphosphokinase (EC 2.7.4.6.) activity was very high and probably equilibrates the UTP–UDP and ATP–ADP couples. Lactose synthetase and hexokinase (EC 2.7.1.1) appeared to catalyse rate-limiting reactions. 4. Large increases were seen of UDP-glucose pyrophosphorylase (5-fold), lactose synthetase A protein (3.8-fold) and α-lactalbumin (28-fold), but not of hexokinase, phosphoglucomutase, UDP-glucose epimerase, nucleoside diphosphokinase or glucose 6-phosphate dehydrogenase (EC 1.1.1.49) activities. 5. It appeared that the increased lactose synthesis was largely accounted for by the increased lactose synthetase A protein activity and α-lactalbumin.

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In-vivo magnetic resonance imaging studies of mammogenesis in non-pregnant goats treated with exogenous steroids

Journal of Dairy Research ◽

10.1017/s0022029900029691 ◽

1991 ◽

Vol 58 (2) ◽

pp. 151-157 ◽

Cited By ~ 15

Author(s):

Paul A. Fowler ◽

Christopher H. Knight ◽

Margaret A. Foster

Keyword(s):

Magnetic Resonance Imaging ◽

Magnetic Resonance ◽

Maximum Size ◽

Mammary Development ◽

Mammary Tissue ◽

Secretory Cells ◽

Resonance Imaging ◽

Secretion Efficiency ◽

Cessation Of Treatment

SummaryMammogenesis and lactation were induced in five multiparous, non-pregnant goats by treatment with oestrogen and progesterone for 11 d, followed by dexamethasone for 3 d. Reserpine was administered during the last 5 d. All five goats lactated, although milk yield was less than had been achieved in previous natural lactations. Mammary development was assessed in vivo, using magnetic resonance imaging. Although parenchyma volume increased by more than 6-fold overall, only 25% of this increase occurred during steroid treatment. Most development took place after the cessation of treatment, when milking commenced. Maximum size was not achieved until week 8 of the induced lactation, and was only 70% of normal parenchyma volume. After 18 weeks lactation the activities of three key milk synthetic enzymes were very similar to values previously found in natural lactations, and secretion efficiency (milk production per unit volume of parenchyma) was also similar to that of natural lactations. We conclude that the lower than normal milk yields were associated with incomplete proliferation of mammary tissue, rather than inadequate differentiation of individual secretory cells.

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The role of the mammary gland during heat stress

10.32469/10355/72201 ◽

2019 ◽

Author(s):

◽

Ricardo Oliveira Rodrigues

Keyword(s):

Blood Flow ◽

Heat Stress ◽

Mammary Gland ◽

Dairy Cows ◽

Prolonged Exposure ◽

Transcriptional Profiling ◽

Mammary Tissue ◽

Secretory Cells ◽

University Of Missouri ◽

Lactating Dairy Cows

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Disruptive effects of climate change, such as increasing environmental temperature, have direct impacts on economic viability and efficiency of food production. In lactating dairy cows, heat stress reduces milk production and alters function of mammary secretory cells, at least partly by disturbing local protein metabolism. We hypothesized that hyperthermia would not only reduce mammary blood flow but would also reduce mammary extraction of nutrients from blood. In addition, we hypothesized that transcriptional profiling of mammary tissue would reveal disruption of cellular homeostasis. Our objective was to determine the effects of hyperthermia on mammary function. More specifically, we aimed to profile mammary blood flow and the changes in mammary transcriptome of heat-stressed lactating dairy cows. We investigated the effects of early and prolonged exposure of lactating dairy cows to hyperthermia by exposing cows to programmed constantly elevated temperature and humidity to induce and maintain body temperature approximately 1[degree]C above normal. Experiments were conducted to evaluate the production responses of hyperthermic lactating dairy cows, to characterize total and nutritive mammary blood flow, and to elucidate the regulation of mammary function during early and prolonged exposure to hyperthermia. Results from these studies established that 1) hyperthermia reduces total and nutritive mammary blood flow, limiting nutrient disappearance across the mammary gland; 2) hyperthermia does not induce shunting of blood away from the gland; 3) hyperthermia affects mammary tissue transcriptome, mainly altering processes associated with ECM and cell adhesion; 4) the effects of exposure to prolonged heat stress on mammary gene expression are distinct from the effects of feed restriction, in lactating dairy cows; and 5) mammary function is reestablished within 8 days after cessation of heat stress.

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Occludin protects secretory cells from ER stress by facilitating SNARE-dependent apical protein exocytosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909731117 ◽

2020 ◽

Vol 117 (9) ◽

pp. 4758-4769 ◽

Cited By ~ 2

Author(s):

Tao Zhou ◽

Yunzhe Lu ◽

Chongshen Xu ◽

Rui Wang ◽

Liye Zhang ◽

...

Keyword(s):

Er Stress ◽

Protein Secretion ◽

Milk Proteins ◽

Late Pregnancy ◽

Mutant Mice ◽

Snare Proteins ◽

Secretory Cells ◽

Tissue Polarity ◽

Pregnancy And Lactation ◽

Dependent Protein

Tight junctions (TJs) are fundamental features of both epithelium and endothelium and are indispensable for vertebrate organ formation and homeostasis. However, mice lackingOccludin(Ocln) develop relatively normally to term. Here we show thatOclnis essential for mammary gland physiology, as mutant mice fail to produce milk. Surprisingly,Oclnnull mammary glands showed intact TJ function and normal epithelial morphogenesis, cell differentiation, and tissue polarity, suggesting thatOclnis not required for these processes. Using single-cell transcriptomics, we identified milk-producing cells (MPCs) and found they were progressively more prone to endoplasmic reticulum (ER) stress as protein production increased exponentially during late pregnancy and lactation. Importantly,Oclnloss in MPCs resulted in greatly heightened ER stress; this in turn led to increased apoptosis and acute shutdown of protein expression, ultimately leading to lactation failure in the mutant mice. We show that the increased ER stress was caused by a secretory failure of milk proteins inOclnnull cells. Consistent with an essential role in protein secretion, Occludin was seen to reside on secretory vesicles and to be bound to SNARE proteins. Taken together, our results demonstrate thatOclnprotects MPCs from ER stress by facilitating SNARE-dependent protein secretion and raise the possibility that other TJ components may participate in functions similar toOcln.

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Cyclic AMP-dependent protein kinase in rat mammary tissue: expression of catalytic and regulatory subunits throughout pregnancy and lactation

Biochemical Journal ◽

10.1042/bj3010807 ◽

1994 ◽

Vol 301 (3) ◽

pp. 807-812 ◽

Cited By ~ 3

Author(s):

R A Gardner ◽

M T Travers ◽

M C Barber ◽

W R Miller ◽

R A Clegg

Keyword(s):

Catalytic Activity ◽

Protein Kinase ◽

Mammary Development ◽

Mammary Tissue ◽

Early Lactation ◽

Dependent Protein Kinase ◽

Regulatory Subunits ◽

C Subunit ◽

Pregnancy And Lactation ◽

Dependent Protein

‘Expressed’ and ‘total’ activities of cyclic AMP-dependent protein kinase (PK-A) were measured in extracts of rat mammary tissue sampled throughout pregnancy and lactation. Expression of the genes encoding the catalytic subunit (C-subunit) isoforms C alpha and C beta was examined by Northern blotting, as a function of mammary development, to determine relative levels of their respective mRNAs. The content of C-subunit protein (all isoforms) was estimated immunochemically and related to levels of C-subunit catalytic activity and of mRNAs. It was found that C-subunit isoform mRNAs are expressed co-ordinately during mammary development and that a marked decline in expression, per cell, at around parturition is paralleled by a fall in ‘total’ PK-A activity. The ‘expressed’ activity of PK-A activity underwent characteristic changes throughout pregnancy and lactation, reaching a peak late in pregnancy. The PK-A activity ratio reached a peak in early lactation. C-subunit protein mass closely parallel ‘total’ PK-A activity throughout pregnancy and lactation, thereby demonstrating the constancy of C-subunit specific catalytic activity during these developmental events. Regulatory subunits (R-subunits) were probed with the photoaffinity label 8-azido-[32P]cAMP. The abundance of R-II as a proportion of total R-subunit increased throughout pregnancy and lactation, and quantitative analysis of the photoaffinity labelling suggested inconstancy in the ratio of R:C subunits, with highest values occurring in late pregnancy/early lactation.

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