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In many tissues, the features of cell nuclei are specific to their differentiated state, notably in terms of the nature and distribution of nuclear compartments and the position of chromosomes and genes. This spatial organization of the nucleus reveals domains that are differentially permissive for gene expression and may constitute an epigenetic mechanism that is involved in maintaining tissue-specific expression profiles. The mammary gland is a complex tissue in which mammary epithelial cells (MECs), which synthesize and secrete milk components, interact with other cell types (myoepithelial cells, adipocytes) and the extracellular matrix. MECs cultures can to some extent recreate cell differentiation in vitro and have been used to follow the development and functional importance of nuclear organization. They have made it possible to show how hormonal stimulation can lead to a remodeling of nuclear domains and the repositioning of genes specific to the mammary gland, such as milk protein genes. By modulating the growth conditions of culture in order to replace cells in a microenvironment similar to that of mammary gland tissue, it should be possible to study the role of this cellular microenvironment in nuclear organization., (© Société de Biologie, 2010.)

Cadherins are transmembrane glycoproteins involved in cell-cell adhesion, signalling, proliferation and differentiation. In this review, we have focused upon in vivo cadherin expression and function in two different biological systems, the mammary gland epithelium and the melanocyte lineage. Development of the mammary gland represents a paradigm of in situ epithelial differentiation and the melanocyte lineage of a model of non-epithelial (or mesenchymal) cell differentiation where cells migrate extensively from their site of origin towards the skin compartment. In the mammary epithelium, the predominantly expressed cadherin is E-cadherin, a cell surface molecule that directs morphogenesis and maintenance of the epithelial structure. In the melanocyte lineage, the expression of a number of cadherins is strictly spatiotemporally regulated during development and adult life. The specific functions mediated by this very dynamic cadherin expression are not yet known and their characterisation represents a challenge for the future.

Using records from the Milk Registration Organization for 254 dairy-goat farms located in western France, the economical impact of mammary gland inflammation on milk yield and composition was measured after 200 days of lactation. This assessment was performed in groups of goats belonging to the same farm and the same breed and having the same parity. Three levels of somatic cell count (SCC) were defined in order to discriminate goats on the basis of the intensity of inflammatory reaction of their udder: < 750,000 cells/mL (CCI1), between 750,000 and 1,750,000 cells/mL (CCI2), > 1,750,000 cells/mL (CCI3). From 404 groups characterized by an average number of 12,4 CCI1 goats and 14,2 (CCI2 + CCI3) goats, the average milk yield of the goats whose SCC was superior to 750,000 cells/mL was inferior to that of the CCI1 goats in 79.2% of the groups. In a sub-sample of 366 groups, the average loss of milk production for the CCI2 goats compared to the CCI1 ones was 55 kg. In an other sub-sample of 41 groups, the milk production average loss of the CCI3 goats reached 132 kg. For all of the 404 groups, (CCI2 + CCI3) goats had a lower fat content of 0.3 g/kg and in contrast a higher protein content of 0.6 g/kg. A second evaluation using a multidimensional variance analysis model in a population of 20796 goats showed the same results.

The bulky production of recombinant proteins can be achieved by procaryotes or eucaryotes cells. Cells from higher eucaryotes may be required when proteins have to be modified post-transcriptionally (glycosylation phosphorylation, cleavage, folding...). Cells from higher vertebrates in culture are used to prepare proteins like human factor VIII and erythropoietin. The use of transgenic organism has been suggested to reach the same goal. Indeed a whole living organism allows a very potent amplification, the number of cells involved in the biosynthesis of the recombinant proteins being very numerous and in the best metabolic conditions. Biological fluids (blood, milk, insect hemolymph, egg white...) and possibly organs from transgenic animals are a priori the best sources of recombinant proteins. Blood is abundant and it is a by-product of slaughter house. Its composition is relatively complex and the circulating recombinant proteins may heavily alter health of animals. Milk is very abundant, its composition is relatively simple, it is poor in proteolytic enzymes and it can be collected easily. Hemolymph from insects is relatively scarce. Egg white will be a possible source of recombinant proteins, when transgenesis has become more accessible in birds. Organs from transgenic animals should be solicited only when a particular cell type is required for the biosynthesis of the recombinant proteins. Milk appears therefore, presently, as the best source of recombinant proteins from transgenic animals. About 15 public and private laboratories try to use these techniques. They consist in preparing vectors containing regulatory regions of one of the milk proteins genes and the coding part (cDNA or gene) of the corresponding proteins to be produced. The transfer of these gene constructs to mouse, rabbit, sheep, goat, pig, shows that these techniques are indeed very promising. A single protein, human alpha 1-antitrypsin produced in milk of transgenic sheep, has presently reached the preparation at an industrial scale. This method has two theoretical limitations: 1) some of the proteins secreted in milk may be not matured as their native counterparts. Experiments carried out so far (about 20 proteins has been produced at an experimental scale) indicate that the mammary cell is able to achieve glycosylation in a correct way; 2) a significant proportion of the recombinant proteins migrate from the alveolar compartment of the mammary gland to blood circulation and they can alter health of lactating animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Seven lactating Lacaune ewes underwent either a total luteectomy on day 19 of pregnancy (D19) (compensated from that stage by a daily progesterone supplementation of 25 mg to ensure embryonic survival; group 1:4 animals) or a control laparotomy (group 2: 3 animals). Intra-jugular injection of 200 micrograms of a synthetic PGF2 alpha analogue (Dinolytic, Upjohn) caused an increase in the intramammary pressure (IMP) and a concomitant rise in oxytocinaemia only in the presence of a corpus luteum, ie in all ewes of groups 1 and 2 before D19 and only in those of group 2 after that stage. These experiments confirm that the corpus luteum, and not the other ovarian compartments, releases oxytocin when prostaglandin F2 alpha is administered.

The expression of casein genes is under the control of several hormones of which the most important are prolactin, glucocorticoids and progesterone. In pseudopregnant or mid-pregnant rabbit having partially developped but inactive mammary gland, prolactin induces casein synthesis. The phenomenon is accompanied by an accumulation of casein mRNAs and by a stimulation of their translation. The accumulation of casein mRNAs results from an acceleration of the transcription of the corresponding genes and from a stabilization of the mRNAs. These prolactin effects are amplified by glucocorticoids which are not per se inducers and they are inhibited by progesterone. The essential action of prolactin and glucocorticoids can be obtained in cultured mammary explants and epithelial cells. This induction is accompanied by a transformation of the mammary cell in which are accumulated ribosomes and membranes involved in milk synthesis and exportation. This transformation is favoured by prolactin and inhibited by progesterone. Hence, the abundant milk secretion is triggered only after parturition when the predominence of progesterone is reversed in favour of prolactin. Prolactin incubated with mammary membranes promotes the formation of a factor capable of accelerating beta-casein gene transcription when added to isolated mammary nuclei. This factor is formed only by lactogen hormones and from prolactin receptor containing membranes. The information contained in the factor seems to be understood only by prolactin target genes. The generation of the factor can be provoked by anti-prolactin receptor antibodies and it is inhibited by tubulin binding drugs such as colchicine. The molecule exhibiting prolactin-like activity has a small molecular weight, it is thermostable and inactivated by trypsin. The stimulation of beta-casein gene transcription is abolished when the factor is incubated with nuclei in the presence of phosphatase inhibitors. These facts suggest that prolactin after its binding to its peripheral receptors triggers the release of a small peptide which migrates to nuclei where it activates the transcription of the prolactin target genes through a dephosphorylation of nuclear proteins. This small peptide is a good candidate to the prolactin intracellular relay.

Development and differentiation of mammary gland are controlled by a large number of hormones. In embryo, foetal androgens induce a partial necrosis of mammary epithelium. This action is mediated by cells of the parenchyma which is the real target of androgens. Estrogens are not true growth factor in normal mammary gland: they deliver a message of unknown chemical nature, which allows growth factors to act. The growth factors of mammary cells are only partially known. Clones of the various mammary cell types have been obtained (fibroblasts, adipocytes, myoepithelial and epithelial cells). These clones are a good tool to determine growth factors specific of each cell type. Under the influence of GH, mammary fibroblasts are transformed into preadipocytes which secrete a growth factor (PGE2) which specifically stimulates multiplication of mammary epithelial cells. Other growth factors induced by oestrogens or prolactin have been identified but their exact role remains unknown. Induction of milk synthesis is under the strict dependency of prolactin. The action of this hormone is strongly stimulated by glucocorticoids and insulin at high concentrations and it is inhibited by progesterone and EGF. Prolactin stimulates transcription of milk protein genes and this stimulation is modulated by glucocorticoids and progesterone. Induction of casein and DNA synthesis by prolactin can be mimicked by polyclonal and monoclonal anti-prolactin receptor antibodies. Collagen is necessary for isolated epithelial mammary cells to respond to prolactin signal. The role of other components of the extracellular matrix (proteoglycans, glycoproteins) is partially known. Specific peripheral markers corresponding to the different cell types and to the various stages of mammary gland development have been identified. All these cellular and molecular parameters are compared in normal and tumor mammary cells to point out possible differences.

The increase in intramammary pressure caused by injecting small amounts of water into the udder artery of anesthetized ewes does not seem to be a nervous reaction induced by the stimulation of sensitive mammary receptors: the deflections remain unchanged after complete denervation of the gland (fig. 4) and after the injection of water at different temperatures (4 to 37 degrees C; fig. 1) or pH's (4.6 to 9.8; fig. 2). Since injecting the same volumes of blood (fig. 3), isotonic NaCl (fig. 5) or glucose (fig. 6) solutions does not cause the intramammary pressure to increase, water effect would appear to result from a change in blood osmotic pressure. Among the substances released when the blood corpuscles or platelets are destroyed, kallikrein and histamine do not seem to play a role. These products induce intramammary pressure to rise (fig. 7, 9) but latency and duration are longer than with water Also, their respective inhibitors, aprotinin and phenergan, always block their effect on intramammary pressure but not that of water. On the other hand, water and bradykinin (fig. 8) produce similar responses, indicating that the latter substance may play a role. We must consider also the possible action of extra-capillary water transfer, modifying ion flux and myoepithelial cell membrane potential or simply inducing the alveolar cells to swell and the acini to flatten. This last hypothesis permits a better interpretation of the pressure decreases noted after injection of hypertonic solutions (figs. 5, 6) since reverse water movements would have the opposite effect.

We have studied the estrogenic action of zeranol and have compared it to the action of estradiol on the uterus--the vagina, the mammary gland and the pituitary gland of the pre-pubescent, castrated female before the opening of the vagina. The weakest dose at which estradiol is active is 0.1 microgram injected for a period of three days : it is capable of opening the vagina on the fourth or fifth day and of developping the uterine glands to the maximum : injected over a period of ten days it provokes a hypertrophy of the pituitary gland : only the mammary gland reacts exclusively to doses other than physiological ones. The estrogenic action of zeranol is weak : 1 mg administered orally for three days is necessary in order to reproduce the same effects of 0,1 microgram of estradiol on the opening and the keratinisation of the vagina and on the stimulation of the uterine epithelium ; its efficacy is greater on the uterine glands (250 micrograms is equivalent to 0.1 microgram) and on the pituitary gland (500 microgram reproduce the hypertrophy provoked by 0,1 microgram). The doses administered are by 100 g of body weight. The uterine glands are more sensitive to stimulation by the estrogens employed (estradiol, zeranol) than is the uterine epithelium. The study of the relation between the estrogenic dose and the response of the uterine weight shows that the gradient of the curve is weaker with zeranol than with estradiol. This observation shows that zeranol behaves as an impeded estrogen.

During suckling, the intramammary pressure and the electrical activity of oxytocinergic cells in the paraventricular nucleus were recorded simultaneously in urethane-anaesthetized Rats. Injection of an oxytocin antagonist (1 microliter d(CH2)5OVT 10(-3) or 10(-4) M) into the 3rd ventricle, inhibited both frequency and amplitude of neurosecretory bursts and milk-ejections. The same effect was observed when the milk-ejection reflex was previously activated by the intraventricular injection of oxytocin (1 microliter--10(-6) M). Thus, oxytocin might regulate the frequency and amplitude of oxytocinergic cells' neurosecretory bursts and could be involved in their synchronism.

The effects of two neuroleptics (pipotiazine and fluphenazine) and five long-acting neuroleptics (pipotiazine undecylenate and palmitate, fluphenazine enanthate and decanoate, fluopentixol decanoate) are tested in the rat, during an observation period of 20 to 40 days following only one injection of compound. The compounds administered at three different and non toxic doses, are showing effects, the intensity and duration of which are different according to the dose and the compound: diestrus of pseudo-gestation or more than 15 days, hypertrophy of mammary gland, decreasing of the uterine weight. Some long-acting neuroleptics are active during more than forty days.

Prolactin and arachidonic acid increase milk casein secretion in mammary gland slices. These effects do not necessitate Ca2+ in the incubation medium. Prolactin does not modify the influx or the efflux of 45Ca2+. The Ca2+ channel blocking agent D600 (6 micrograms/ml) decreases the stimulatory effect of prolactin on casein secretion, but does not interfere in the stimulatory effect of arachidonic acid. The calmodulin inhibitor trifluoperazine (100 microM) inhibits stimulation of casein secretion by both prolactin and arachidonic acid. From these data, it is concluded that a flow of Ca2+ from the outside into the cell is not a requisite for the stimulation of casein secretion. However, stimulation by prolactin, but not stimulation by arachidonic acid, requires Ca2+ movement through calcium pathways. Intracellular transport of Ca2+ seems necessary for the stimulation of secretion.

Maternal behaviour and PRL response to mammary stimulation have been studied in three groups of 20 Friesian cows each, held according to three different systems of management. In the 1st group (group AL), the cows suckled 3 alien calves and were all kept in one pen after a fostering period of two weeks. In the second group (group AE), the cows suckled 3 alien calves twice a day when tethered. In this group, the fostering procedure was limited to 18 h. In the 3rd group (group T), the cows were separated from their calves just after birth and then milked twice a day. On day 45 after calving, mother-young relationships were observed in the three groups for one day, the animals all being kept with familiar or alien calves on this occasion. Then, on day 112 postpartum, the PRL response to suckling (groups AL and AE) or to milking (group T) was studied in 4 cows of each group. Group AL cows were maternal and selective. They preferentially suckled their foster calves rather than alien ones and suckling was carried out in parallel-inverse position. Group AE cows were also maternal but the relationships were not selective and suckling in parallel-inverse position was less frequent. Finally, most milked cows did not allow the calves to suck. A comparison of PRL response to mammary stimulation showed that the response of milked cows (group T) to milking was low whereas suckling by an alien calf in group AL did not lead to a PRL response; the presence of the 3 foster calves was needed to induce this response. These results indicate that breeding conditions after calving influence mother-young relationships and suggest that maternal behaviour is a modulating factor of PRL response to suckling.

The lysosomotropic agents, ammonium chloride and chloroquine, added to the culture medium of pseudopregnant Rabbit mammary gland, did not inhibit the initiation of casein synthesis by prolactin. By contrast, they considerably reduced the down-regulation of prolacting receptors. Converse-y, colchicine totally blocked the lactogenic action of prolactin without altering the down-regulation of the receptor. Cytochalasin B inhibited only partly the lactogenic action of prolactin while it has no effect on the down-regulation of the receptor. These data suggest that the degradation of the prolactin-receptor complex in lysosome is not a compulsory step in the mechanism of prolactin action. The integrity of microtubules but not of microfilaments is required for prolactin to initiate casein synthesis. These elements of the cytoskeleton are not strictly involved in the down-regulation of the receptor.

The influence on the pituitary hypothalamic system of Sultopride are different of those of a chemically related compound, Sulpiride. In the mammary gland the development of the acini is associated with an increase of the connective tissue. The processus of secretion, lactogenesis is quiet not stimulated. The changes on the anterior lobe of the pituitary involve the carmino and eosinophilic cells. This modification seems to reflect a stimulation of the secretion of LT and an inhibition of the release of STH. In the juvenile rat, Sultopride determines an inhibition of the somatic growth.

The intrajugular administration of 200 micrograms of Dinolytic (tromethamine salt of PGF2 alpha) to lactating ewes caused an intramammary pressure (IMP) increase only during the luteal phase of the sexual cycle (group A). The increase in progesteronemia, induced by three daily injections of 2.5 micrograms of LH-RH, did not modify response amplitude (group B). On the other hand, bilateral ovariectomy (groupe C) led to the suppression of those responses, but supplementary oestroprogestative (table 1) did not re-establish them. It is probable that the milk ejection caused by PGF2 alpha resulted from a release of oxytocin by the corpus luteum. The ineffectiveness of the exogenous and endogenous reinforcement of progesterone therefore suggests that this hormone plays no part in the putative control of hypothalamic receptivity to prostaglandins.