scholarly journals Identification of reference genes for RT-qPCR in ovine mammary tissue during late pregnancy and lactation and in response to maternal nutritional programming

2014 ◽  
Vol 46 (15) ◽  
pp. 560-570 ◽  
Author(s):  
A. M. Paten ◽  
S. J. Pain ◽  
S. W. Peterson ◽  
H. T. Blair ◽  
P. R. Kenyon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Reference Genes ◽  
Physiological State ◽  
Late Pregnancy ◽  
Mammary Tissue ◽  
Qpcr Data ◽  
Maternal Programming ◽  
Pregnancy And Lactation ◽  
Measure Gene Expression

The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse-transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes ( PRPF3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analyzing gene expression in individual animals ( SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland.

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

2005 ◽  
Vol 289 (4) ◽  
pp. E634-E642 ◽  
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.

Ultrasonographic assessment of the feline mammary gland

2008 ◽  
Vol 10 (5) ◽  
pp. 466-471 ◽  
Author(s):  
Rita Payan-Carreira ◽  
Ana C. Martins-Bessa
Keyword(s):  
Mammary Gland ◽  
Diagnostic Tool ◽  
Late Pregnancy ◽  
Mammary Glands ◽  
Mammary Tissue ◽  
Physiological Changes ◽  
Linear Transducer ◽  
Pregnancy And Lactation ◽  
Ultrasonographic Assessment ◽  
Ultrasonographic Appearance

The aim of this study is to characterise the feline mammary echotexture using B-mode ultrasonography, which is not routinely used to examine the feline mammary gland. Using a 5–9 MHz linear transducer the ultrasonographic appearance of non-stimulated and stimulated mammary glands was determined in 35 mature intact non-pregnant, pregnant and lactating queens aged from 16 months to 8 years. In intact non-pregnant queens, mammary glands are fairly underdeveloped and on the ultrasonograms they appear with a regular hypoechoic texture and generally show a thickness of less than 2.0 mm. The stimulated mammary tissue typically presents a more hyperechoic appearance compared to the non-stimulated gland and a fine granular echotexture. Maximum echogenicity of the mammary gland is reached during lactation. In late pregnancy, the mammary glands reach 6–9 mm in thickness. During lactation, the size of the glands depends on the existence of a suckling stimulus, with the suckled glands reaching about 11 mm in thickness. Ductal structures can only be imaged during late pregnancy and lactation. Ultrasonographic evaluation of the feline mammary gland can become a valuable diagnostic tool to characterise physiological changes and may further contribute to a better characterisation of diseased mammary tissue.

Developmental expression of pIgR gene in sheep mammary gland and hormonal regulation

2002 ◽  
Vol 69 (1) ◽  
pp. 13-26 ◽  
Author(s):  
AURORE RINCHEV-ALARNOLD ◽  
LUCETTE BELAIR ◽  
JEAN DJIANE
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Hormonal Regulation ◽  
Mrna Level ◽  
Immunohistochemical Analysis ◽  
Developmental Expression ◽  
Secretory Iga ◽  
Mrna Levels ◽  
Immunoglobulin Receptor ◽  
Pregnancy And Lactation

Secretory IgA found in external secretions are constituted by polymeric IgA (pIgA) bound to the extra-cellular part of the polymeric immunoglobulin receptor (pIgR). The receptor mediates transcytosis of pIgA across epithelial cells. The aim of the present study was to analyse the evolution of pIgR expression in the sheep mammary gland during the development of the mammary gland and to analyse its hormonal regulation. Gene expression of the pIgR was analysed in sheep mammary gland during pregnancy and lactation. By Northern Blot analysis, we observed that low levels of pIgR mRNA are expressed until day 70 of pregnancy. Accumulation of pIgR mRNA started during the third part of pregnancy and intensified 3 d after parturition to reach highest levels during established lactation (day 70). In situ hybridization analysis was used to confirm the increase in pIgR gene expression per mammary epithelial cell. In order to examine the hormonal regulation of the pIgR expression, virgin ewes were hormonally treated. Treatment with oestradiol and progesterone increased pIgR mRNA levels slightly. Subsequent addition of glucocorticoids induced a significant accumulation of pIgR mRNA in the mammary gland of the treated animals. Immunohistochemical analysis was performed to verify that the increase of pIgR mRNA level was associated with enhancement of the pIgR protein in mammary cells. No increase of pIgR mRNA levels were observed if PRL secretion was blocked by bromocryptine injections throughout the hormonal procedure. In conclusion, the present experiments suggest that the enhancement of pIgR levels during lactation result from combined effects of both prolactin and glucocorticoids.

scholarly journals Has the mammary gland a protective mechanism against overexposure to triiodothyronine during the peripartum period? The prolactin pulse down-regulates mammary type I deiodinase responsiveness to norepinephrine

2004 ◽  
Vol 183 (2) ◽  
pp. 267-277 ◽  
Author(s):  
B Anguiano ◽  
R Rojas-Huidobro ◽  
G Delgado ◽  
C Aceves
Keyword(s):  
Mammary Gland ◽  
Critical Role ◽  
Alveolar Epithelium ◽  
Late Pregnancy ◽  
Protective Mechanism ◽  
Type I ◽  
Key Factor ◽  
Pregnancy And Lactation ◽  
Contrary Effect ◽  
Peripartum Period

Peripartum is a crucial period for mammary gland final differentiation and the onset of lactation. Although the ‘trigger’ for lactogenesis depends on several hormones, a key factor is the peripartum prolactin (PRL) pulse whose deletion results in a failure to initiate milk production. Other hormones having a critical role during this period but exerting a contrary effect are the thyronines. A transitory hypothyroidism occurs at peripartum in serum and several other extrathyroidal tissues, whereas the induction of hyperthyroidism during late pregnancy is associated with the absence of lactation after delivery. We analyzed the mammary gland during pregnancy and lactation for: (a) the type and amount of thyroid receptors (TRs), (b) the local triiodothyronine (T3) generation catalyzed by type I deiodinase (Dio1), (c) the Dio1 response to norepinephrine (NE) and (d) the effect on Dio1 and TRs of blocking the PRL pulse at peripartum. Our data showed that during pregnancy the mammary gland contains Dio1 in low amounts associated with the highest expression of TRα1; whereas during lactation the gland shows high levels of both Dio1 and TRα1. However, at peripartum, both TRs and Dio1 decrease, and Dio1 becomes refractory to NE. This refractoriness disappears when the PRL pulse is blocked by the dopamine agonist bromocriptine. This blockade is also accompanied by a significant decrease in cyclin D1 expression. Our data suggested that the peripartum PRL pulse is part of a protective mechanism against precocious differentiation and/or premature involution of the alveolar epithelium due to T3 overexposure.

Uteroplacental insufficiency alters the mammary gland response to lactogenic hormones in vitro

2008 ◽  
Vol 20 (4) ◽  
pp. 460 ◽  
Author(s):  
Rachael O'Dowd ◽  
Mary E. Wlodek ◽  
Kevin R. Nicholas
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Postnatal Growth ◽  
Mammary Development ◽  
Mammary Tissue ◽  
Sham Surgery ◽  
Uterine Vessel ◽  
Lactogenic Hormones ◽  
Vessel Ligation

Adequate mammary development and coordinated actions of lactogenic hormones are essential for the initiation of lactation. Pregnancies compromised by uteroplacental insufficiency impair mammary development and lactation, further slowing postnatal growth. It is not known whether the initiation of lactation or galactopoesis is compromised. Uteroplacental insufficiency induced in rats by bilateral uterine vessel ligation (Restricted) or sham surgery (Control) on Day 18 of gestation preceded collection of mammary tissue on Day 20 of pregnancy. Mammary explants were cultured with combinations of insulin, cortisol and prolactin and analysed for α-lactalbumin and β-casein gene expression. Mammary tissue from late pregnant Restricted rats had elevated α-lactalbumin, but not β-casein, mRNA, which is consistent with premature lactogenesis resulting from an early decline in peripheral maternal progesterone. Explants from Restricted rats were more responsive to hormone stimulation after 3 days in culture, indicating that compromised galactopoesis, not lactogenesis, most likely leads to the reduced growth of suckled pups.

XXIV.—The Innervation of the Human Quiescent Nipple, with Notes on Pigmentation, Erection, and Hyperneury

1949 ◽  
Vol 61 (3) ◽  
pp. 699-717 ◽  
Author(s):  
E. P. Cathcart ◽  
F. W. Gairns ◽  
H. S. D. Garven
Keyword(s):  
Mammary Gland ◽  
Considerable Change ◽  
The Other ◽  
Mammary Tissue ◽  
Histological Structure ◽  
Nerve Fibres ◽  
Histological Studies ◽  
Pregnancy And Lactation ◽  
To Receive ◽  
Stimulation Of

It has been known from antiquity that involution of the uterus is aided by putting the child to the breast, and the work of Moir (1933) has demonstrated conclusively that suckling brings about waves of contraction of the puerperal uterus. Since suckling must necessarily involve stimulation of the nipple, it seemed of interest to know what sensory apparatus is present in the nipple to receive these stimuli. While histological studies of the mammary gland itself have been numerous, only slight attention has been paid to the histological structure of the areola and the nipple.It must be appreciated from the outset that the present study is devoted to the innervation of the nipple in the quiescent breast. There may be considerable change during pregnancy and lactation, not only in the size of the organ and its epithelium but also in the other structures. It has been shown that there is an increase in the number of nerve-fibres in the actively secreting mammary tissue, and it is possible that there is an increase in the nerve-structures of the nipple also. The richness of the innervation in the quiescent nipple certainly makes a further study of these nerve-structures during pregnancy and lactation of great interest.

Ruminants express a prolactin receptor of Mr 33 000—36 000 in the mammary gland throughout pregnancy and lactation

1993 ◽  
Vol 139 (1) ◽  
pp. 37-49 ◽  
Author(s):  
J. J. Smith ◽  
A. V. Capuco ◽  
I. H. Mather ◽  
B. K. Vonderhaar
Keyword(s):  
Mammary Gland ◽  
Developmental Stages ◽  
Developmental Regulation ◽  
Prolactin Receptor ◽  
Binding Activity ◽  
Receptor Type ◽  
Mammary Tissue ◽  
Cross Linking ◽  
Developmental Variation ◽  
Pregnancy And Lactation

ABSTRACT Developmental variation in the expression of the prolactin receptor in the ruminant mammary gland was investigated. Affinity chromatography revealed that bovine prolactin and human GH each bound to the same mammary gland proteins, yielding fractions enriched in binding activity and a protein of Mr 36 000, assumed to be a bovine prolactin receptor. Affinity cross-linking of 125I-labelled human GH to mammary microsomes confirmed that the Mr 36 000 protein was a bovine prolactin receptor. Binding assays of receptors in microsomes from the mammary tissue of cows and ewes at various stages of the lactational/reproductive cycle indicated developmental regulation of receptor concentration, but not receptor type, as no other bovine prolactin receptor type was detected by affinity cross-linking. These results suggest that differences in the response to prolactin in the mammary gland at various developmental stages in ruminants are not due to the expression of different forms of the prolactin receptor, and the lack of a prolactin effect on established lactation in ruminants is not due to the absence of the Mr 36 000 form of the prolactin receptor. Journal of Endocrinology (1993) 139, 37–49

scholarly journals Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland

1999 ◽  
Vol 112 (11) ◽  
pp. 1771-1783 ◽  
Author(s):  
A.D. Metcalfe ◽  
A. Gilmore ◽  
T. Klinowska ◽  
J. Oliver ◽  
A.J. Valentijn ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
De Novo ◽  
Mammary Epithelial Cells ◽  
Expression Profiles ◽  
Mammary Epithelial ◽  
Mammary Tissue ◽  
Pregnancy And Lactation

Epithelial cells within the mammary gland undergo developmental programmes of proliferation and apoptosis during the pregnancy cycle. After weaning, secretory epithelial cells are removed by apoptosis. To determine whether members of the Bcl-2 gene family could be involved in regulating this process, we have examined whether changes in their expression occur during this developmental apoptotic program in vivo. Bax and Bcl-x were evenly expressed throughout development. However, expression of Bak and Bad was increased during late pregnancy and lactation, and the proteins were present during the time of maximal apoptotic involution. Thereafter, their levels declined. In contrast, Bcl-w was expressed in pregnancy and lactation but was downregulated at the onset of apoptosis. Bcl-2 was not detected in lactating or early involuting mammary gland. Thus, the pro-apoptotic proteins Bax, Bak and Bad, as well as the death-suppressors Bcl-x, Bcl-2 and Bcl-w, are synthesised in mouse mammary gland, and dynamic changes in the expression profiles of these proteins occurs during development. To determine if changes in Bak and Bcl-w expression could regulate mammary apoptosis, their effect on cultured mouse mammary epithelial cells was examined in transient transfection assays. Enforced expression of Bak induced rapid mammary apoptosis, which could be suppressed by coexpression of Bcl-w. In extracts of mammary tissue in vivo, Bak heterodimerized with Bcl-x whereas Bax associated with Bcl-w, but Bak/Bcl-w heterodimers were not detected. Thus, Bak and Bcl-w may regulate cell death through independent pathways. These results support a model in which mammary epithelial cells are primed for apoptosis during the transition from pregnancy to lactation by de novo expression of the death effectors Bak and Bad. It is suggested that these proteins are prevented from triggering apoptosis by anti-apoptotic Bcl-2 family proteins until involution, when the levels of Bcl-w decline. Our study provides evidence that regulated changes in the expression of cell death genes may contribute to the developmental control of mammary apoptosis.

scholarly journals Identification of reference genes for RT-qPCR data normalisation in aging studies

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Lourdes González-Bermúdez ◽  
Teresa Anglada ◽  
Anna Genescà ◽  
Marta Martín ◽  
Mariona Terradas
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Levels ◽  
Qpcr Data ◽  
Age Related ◽  
Specific Subset ◽  
Aging Studies

Abstract Aging is associated with changes in gene expression levels that affect cellular functions and predispose to age-related diseases. The use of candidate genes whose expression remains stable during aging is required to correctly address the age-associated variations in expression levels. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a powerful approach for sensitive gene expression analysis. Reliable RT-qPCR assays rely on the normalisation of the results to stable reference genes. Taken these data together, here we evaluated the expression stability of eight frequently used reference genes in three aging models: oncogene-induced senescence (OIS), in vitro and in vivo aging. Using NormFinder and geNorm algorithms, we identified that the most stable reference gene pairs were PUM1 and TBP in OIS, GUSB and PUM1 for in vitro aging and GUSB and OAZ1 for in vivo aging. To validate these candidates, we used them to normalise the expression data of CDKN1A, APOD and TFRC genes, whose expression is known to be affected during OIS, in vitro and in vivo aging. This study demonstrates that accurate normalisation of RT-qPCR data is crucial in aging research and provides a specific subset of stable reference genes for future aging studies.

Localization and expression of BCAT during pregnancy and lactation in the rat mammary gland

2001 ◽  
Vol 280 (3) ◽  
pp. E480-E488 ◽  
Author(s):  
Armando R. Tovar ◽  
Enrique Becerril ◽  
Rogelio Hernández-Pando ◽  
Gabriel López ◽  
Agus Suryawan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Milk Production ◽  
Acinar Cells ◽  
Progressive Increase ◽  
Female Rats ◽  
Specific Staining ◽  
Pregnancy And Lactation ◽  
Branched Chain ◽  
Immunohistochemical Studies

During lactation, branched-chain aminotransferase (BCAT) gene expression increases in the mammary gland. To determine the cell type and whether this induction is present only during lactation, female rats were randomly assigned to one of three experimental groups: pregnancy, lactation, or postweaning. Mammary gland BCAT activity during the first days of pregnancy was similar to that of virgin rats, increasing significantly from day 16 to the last day of pregnancy. Maximal BCAT activity occurred on day 12 of lactation. During postweaning, BCAT activity decreased rapidly to values close to those observed in virgin rats. Analyses by Western and Northern blot revealed that changes in enzyme activity were accompanied by parallel changes in the amount of enzyme and its mRNA. Immunohistochemical studies of the mammary gland showed a progressive increase in mitochondrial BCAT (mBCAT)-specific staining of the epithelial acinar cells during lactation, reaching high levels by day 12. Immunoreactivity decreased rapidly after weaning. There was a significant correlation between total BCAT activity and milk production. These results indicate that the pattern of mBCAT gene expression follows lactogenesis stages I and II and is restricted to the milk-producing epithelial acinar cells. Furthermore, BCAT activity is associated with milk production in the mammary gland during lactation.

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