Regulation of casein messenger RNA during the development of the rat mammary gland

Rosen, Jeffrey M.; Woo, Savio L. C.; Comstock, John P.

doi:10.1021/bi00684a016

Cited by 160 publications

(75 citation statements)

References 25 publications

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“…We have used both the method of translation in a reticulocyte-lysate and hybridization to cDNA to assay for the presence of casein mRNA. In agreement with the results of recent experiments in the rat [12], we have found casein mRNA appearing in the rabbit mammary gland well before the onset of lactogenesis. Furthermore, the sensitivity of the hybridization technique has enabled us to detect casein mRNA in the mammary gland of virgin animals, indicating that there is a low level of transcription of casein genes in undeveloped tissue.…”

supporting

confidence: 92%

“…As a result, it has been possible to detect casein mRNA in mammary glands of virgin animals and also during the very early stages of pregnancy. A similar result has been obtained in studies on the rat where significant amounts of casein mRNA were detectable, by translation in a wheat germ extract, during the early and middle stages of pregnancy, well before the onset of lactogenesis [12]. These authors however, did not attempt to isolate mRNA from polysomes and it was therefore not possible to determine from their results whether the mRNA synthesized prior to lactation was actively translated or stored in the cell.…”

Section: (A-a)supporting

confidence: 56%

“…The concentration of casein mRNA increased more than 150-fold between the 5th and 29th day of pregnancy in the rabbit whereas there was only a 2-fold increase between the 5th and 20th day in the rat [12]. This perhaps reflects the seemingly premature secretion of milk in the rabbit which begins 9 days before parturition whereas substantial secretion in the rat is first observed the day before pregnancy terminates [22].…”

Section: (A-a)mentioning

confidence: 99%

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Studies on the Synthesis of Casein Messenger RNA during Pregnancy in the Rabbit

Shuster

Houdebiné²,

Gaye³

1976

European Journal of Biochemistry

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The quantity of casein mRNA in the rabbit mammary gland was assayed during the course of pregnancy, by translation of casein mRNA in a reticulocyte lysate and by hybridization to DNA complementary to purified casein mRNA. Both methods indicated that the major increases in the concentration of casein mRNA in both polysomal and total cellular RNA occurred between the 18th and 25th day of pregnancy. The change in casein mRNA concentration during this period coincided with a sharp rise in casein synthesis in mammary gland explants suggesting that the levels of casein mRNA determine the rate of casein synthesis in the mammary gland.The sensitivity of the hybridization assay made it possible to detect the presence of casein mRNA in virgin animals and during the very early stages of pregnancy. At day 5, casein mRNA was found associated with polysomes indicating that there was probably some casein synthesis at this early stage of gestation. These results suggest that the hormones controlling lactogenesis in the rabbit may function by augmenting the rate of casein mRNA synthesis rather than initiating transcription of previously inactive genes.Previous studies on the growth of the mammary gland in pregnant rabbits have shown that significant increases in both wet weight and nucleic acid content occur between days 16 and 24 or pregnancy [I-51. It is also during this period that lactose synthetase activity is detected in the mammary gland and the synthesis and secretion of milk is first observed [5 -71.When considered in combination, these results suggested that the genes directing the synthesis of milk proteins were not actively transcribed until mid-pregnancy. At that time, they would presumably be switched on in response to a combination of the same hormonal influences which were responsible for the growth and maturation of the mammary gland [2,8 -111. In this report, we have tested this assumption by examining the content of casein mRNA in rabbit mammary gland during the entire course of pregnancy. We have used both the method of translation in a reticulocyte-lysate and hybridization to cDNA to assay for the presence of casein mRNA. In agreement with the results of recent experiments in the rat [12], we have found casein mRNA appearing in the rabbit mammary gland well before the onset of lactogenesis. Furthermore, the sensitivity of the hybridization technique has enabled us to detect casein mRNA in the mammary gland of virgin animals, indicating that there is a low level of transcription of casein genes in undeveloped tissue. We will present evidence that the casein mRNA detected in mammary glands during the first few days of pregnancy is found associated with a polysomal fraction indicating that it is translated even during the very early stages of gestation. MATERIALS AND METHODS AnimalsPrimiparous rabbits were used. The day of coitus was counted as day 0 of pregnancy. Animals were sacrified by exsanguination and the mammary glands were immediately excised and frozen at -30 "C. Four animals were used in each group...

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supporting

confidence: 92%

Section: (A-a)supporting

confidence: 56%

Section: (A-a)mentioning

confidence: 99%

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Studies on the Synthesis of Casein Messenger RNA during Pregnancy in the Rabbit

Shuster

Houdebiné²,

Gaye³

1976

European Journal of Biochemistry

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“…The hormones insulin (bovine), growth hormone (bovine), prolactin (ovine), progesterone, and estradiol-17f3 were from Calbiochem, La Jolla, CA, and cortisol was from Sigma Chemical Co., St. Louis, MO. Iodine-125 (125I), 17 Ci/mg of I (Nal in 0.1 M NaOH), was Morphogenesis and Functional Differentiation. Fig.…”

Section: Methodsmentioning

confidence: 99%

Hormone-inducible casein messenger RNA in a serum-free organ culture of whole mammary gland

Terry

Banerjee

Lui

1977

Proc. Natl. Acad. Sci. U.S.A.

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The whole second thoracic mammary gland of estradiol-17fi + progesterone primed 3-to 4-week-old BALB/c femalemice was induced to pregnancy-like lobulo-ilveolar morphogenesis after 6-day cultivation in a serum-free culture medium containing a "growth promoting" hormone mixture, insulin + prolactin + growth hormone (somatotropin) + estra- Mammary cells responding to appropriate hormones in a serum-free, chemically defined medium thus provide a favorable system for studying the multiple hormone interactions regulating production of the milk protein. The specific protein synthesis associated with most endocrine-dependent systems is believed to be regulated by hormonal modification of mRNA population in the target tissue (5, 6). Testing the biological properties of hormone-inducible mRNA requires a direct assay system. Previously we have reported that casein mRNA of the lactating mammary gland of the mouse is faithfully translated in a heterologous cell-free protein synthesis system, derived from ascites tumor cell ribosomes, rabbit reticulocyte factors, and tRNA (7). This report presents the results of our studies on cell-free translation of casein mRNA induced by lactogenic hormones in a serum-free organ culture of the whole mammary gland obtained from an immature female mouse. MATERIALS AND METHODSOrgan Culture. As a prerequisite for the organ culture procedure (3) 3-to 4-week-old BALB/c female mice were primed for 9 days by daily injections (subcutaneous) of 1 ,ug of estradiol-17fl (E) and 1 mg of progesterone (P) in 0.9% saline suspension. The entire second thoracic mammary gland was excised under sterile conditions and cultivated in Falcon plastic culture dishes (60 X 15 mm) containing chemically defined Waymouth's (8) medium (MB752/1 supplemented with Lglutamine (350,gg/ml), penicilin (35 ,g/ml), and different combinations of hormones. The incubation was carried out at 370 in an atmosphere of 95% 02 + 5% CO2 according to the standard procedure (9). The method of organ culture of the whole mammary gland of the mouse has been described in detail (4, 10). For morphological studies the glands were fixed in a mixture of acetic acid and ethanol (1:3) and for biochemical analysis glands were frozen in liquid nitrogen and stored at -80°.Indirect Radioimmunoassay. The mammary tissue was homogenized in a buffer containing 10 mM NaPO4 + 15 mM NaCl (pH = 7.5). The homogenate was centrifuged at 100,000 X g for 1 hr at 4°. Different concentrations of protein (11) in the resulting Sloo supernate were assayed for presence of casein by an indirect radioimmunoassay using 125I-labeled mouse milk casein and casein antiserum, as previously described (12).RNA Extraction. Frozen mammary tissue was pulverized in a mortar and pestle cooled by liquid nitrogen. The tissue powder was then homogenized in a power-driven glass tissue grinder containing six volumes of buffer A (13) and the RNA was extracted by phenol/chloroform, followed by high-salt wash as described by Palmiter (13). According to Palmiter, for translational assays ...

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“…Female Wistar rats under various physiological conditions were killed by cervical dislocation, the abdominal-inguinal mammary glands were removed, and stored at -80°C. Total RNA, used to quantify and to purify C2-casein mRNA, was isolated as described by Rosen et al 24 ) The lactating (5 ~8 day) mammary glands were used for mRNA purification. At each step of purification C2-casein mRNA activity was measured in a wheat germ translation assay system using specific antiserum.…”

Section: Methodsmentioning

confidence: 99%