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OBJECTIVE: Heparan sulfates (HS), the polysaccharide side chains of HS proteoglycans, differ in structure and composition of sulfated domains among various tissue types, resulting in selective protein binding. HS proteoglycans on bone marrow endothelial cells (BMEC) could contribute to tissue specificity of the bone marrow endothelium and play a role in the presentation of chemokines such as stromal cell-derived factor-1 (SDF-1) and adhesion of hematopoietic progenitor cells after stem cell transplantations. We characterized differences in HS structure and SDF-1 binding between BMEC and human umbilical vein endothelial cells (HUVEC).MATERIALS AND METHODS: Expression of HS proteoglycans on human bone marrow microvessels was investigated by immunohistochemical staining. Comparison of three human BMEC cell lines with HUVEC and an HUVEC cell line was studied by flow cytometry using antibodies against different epitopes of the HS polysaccharide chain. HS proteoglycans were biochemically characterized after isolation from metabolically labeled cultures of the BMEC cell line 4LHBMEC and HUVEC. Binding of radiolabeled SDF-1 to 4LHBMEC and HUVEC and competition with heparins were investigated.RESULTS: Bone marrow microvessels constitutively expressed HS proteoglycans. Flow cytometric experiments showed differences in HS chain composition between BMEC and HUVEC. Biochemical characterization revealed more O-sulfation of the N-sulfated domains present in cell-associated HS glycosaminoglycans in 4LHBMEC compared to HUVEC. Binding experiments showed that 4LHBMEC bound more 125[I]-SDF-1 per cell than HUVEC. This could be inhibited largely by heparin and O-sulfated heparin and to a lesser extent by N-sulfated heparin.CONCLUSIONS: Cellular HS from BMEC differs in composition from HUVEC. We postulate that the presence of highly sulfated domains in the HS chains from BMEC contributes to tissue specificity of bone marrow endothelium in which HS may be involved in SDF-1 presentation and adhesion of hematopoietic progenitor cells.

L’expression du génome de l’embryon a été étudiée en utilisant des lapins dont la lignée germinale a été enrichie avec un gène marqueur associé au promoteur du gène tk du virus de l’herpès, et à des séquences régulatrices dérivées d’introns et d’exons du gène humain hprt et de la séquence F101 du polyome. Cette construction, nommée IIPytl2LacZ, s’exprime dans les cellules de l’embryon transgénique collecté au stade une cellule et maintenu en culture. Lorsqu’il est d’origine paternelle, le transgène ne s’exprime in vitro qu’à partir du stade 8 cellules alors que d’origine maternelle il s’exprime à partir du stade 16 cellules. Ainsi, in vitro, l’expression du génome est réprimée pendant les premiers stades de développement de l’embryon de lapin. La répression du génome d’origine maternelle a une durée plus longue que celle du génome d’origine paternelle ; la différence correspond au temps nécessaire à la réalisation un cycle cellulaire supplémentaire. La présence d’aphidicoline, un inhibiteur de synthèse de l’ADN, dès le début de la culture s’oppose au passage de l’embryon du stade 2 au stade 4 cellules. Dans ces conditions, le transgène n’est plus exprimé quelle que soit l’origine germinale du gène. L’addition plus tardive de l’aphidicoline dans le milieu de culture, qui permet d’arrêter le développement de l’embryon au stade 4 cellules, réduit encore significativement l’expression du génome d’origine paternelle ou maternelle. Ces résultats montrent que la transcription devient possible seulement après la réalisation des phases de synthèse de l’ADN, ce qui suggère qu’une réoganisation fonctionnelle du génome de l’embryon est nécessaire pour la levée de la répression de la transcription., Delouis Claude, Forlani Sylvie, Jule Sophia. Répression du génome de l’embryon : comment, pourquoi ?. In: Bulletin de l'Académie Vétérinaire de France tome 151 n°2, 1998. pp. 161-169.

Because male ovine embryos develop faster than female embryos, the transcription of SRY and ZFY, two genes located on the Y chromosome, was examined in preimplantation stages using the reverse transcriptase polymerase chain reaction (RT-PCR). RNA was extracted from pools of ovine embryos matured and fertilized in vitro then cultured in synthetic oviduct fluid medium and recovered from 24 to 207 hr post-insemination (two-cell up to hatched blastocyst stage). Since primers used to amplify ZFY also amplify the homologue ZFX, located on the X chromosome, transcripts were differentiated by digestion with restriction enzymes. ZFY and ZFX transcripts were present in all stages examined following RT-PCR, whereas transcripts for SRY were undetectable in all investigated stages following either RT nested PCR or Southern analysis. The presence of ZFY transcripts suggests that Y chromosome is transcriptionally active during early ovine preimplantation development. The possible relationship between a faster growth of male embryos and the transcription of Y-linked genes at early stages of development is discussed.

The ability to transfer genes into cells from different species with murine recombinant retroviruses was evaluated with the SVnls LacZ reporter gene. Mouse and cat packaging cell lines can be used to transfer amphotropic pseudotype, in human, mouse, cat, rabbit, sheep, horse and beef cells and with a very low efficiency in pig and avian cells. Xenotropic pseudotype recombinant retroviruses, produced in cat and rabbit packaging cell lines, transferred genes with the same efficiency as amphotropic retroviruses in human, cat, rabbit and sheep cells. In contrast to amphotropic retroviruses, xenotropic retroviruses infect beef, pig and horse cells with a high efficiency. These results emphasize the need to determine carefully the producer cell line (the type of helper virus and the species origin of the cell) for efficient transfer of genes in cells and embryos.

Invasive cytotrophoblasts play a key role in the development of human placenta and is therefore essential for subsequent development of the embryo. Human implantation is characterized by a major trophoblastic invasion that offers a unique model of a controlled and oriented tumor-like process. The ligand-activated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) modulates cell growth and differentiation and might be therefore considered as a tumor suppressor. We have recently reported that PPARgamma, in synergy with its dimerization partner retinoid X receptor (RXR)alpha, controls the invasion of human primary cytotrophoblasts. Because these cells are unable to replicate in culture, we have, in the present study, transformed these primary cells with the simian virus 40 large T antigen for studying the role of PPARgamma in cell invasion process. Our results show that the cell line human invasive proliferative extravillous cytotrophoblast (HIPEC) 65 expressed markers of human invasive primary cytotrophoblast as determined by immunocytochemistry, immunobloting and real-time RT-PCR, and were highly invasive in vitro. We have next studied the role of PPARgamma/RXRalpha heterodimers in cell proliferation and invasion. Our results show that PPARgamma and RXRalpha are co-expressed by HIPEC 65 and that, as commonly observed, activation of PPARgamma/RXRalpha heterodimers with the specific PPARgamma agonist rosiglitazone induced lipid droplet accumulation as revealed by oil red O staining. Treatment with rosiglitazone or with the natural PPARgamma agonist 15-deoxy-delta-(12,14) PGJ2 did not modify cell growth, but interestingly, activation of PPARgamma by this synthetic (rosiglitazone) or natural (15d-PGJ2) ligand markedly inhibited cell invasion in a concentration-dependent manner. Finally, we showed that other potential natural PPARgamma ligand such as oxidized-but not native-low-density lipoprotein inhibited cell invasion. This proliferative and invasive human cytotrophoblast cell line from extravillous origin provides a new tool for studying specifically the role of PPARgamma in the control of cell invasion.

The objective of this study was to generate immortalized Sertoli cell lines from prepubertal lamb testes to facilitate investigations during the course of testicular differentiation. The Sertoli cells were enzymatically isolated and immortalized by transfection, with the sequences coding for the SV40 large T-antigen fused downstream of regulatory elements from the human vimentin gene. The different cell lines were positively stained with antibodies to vimentin and transferrin, in agreement with their Sertoli origin. Reverse transcriptase polymerase chain reaction was used to analyze the specific expression of molecular markers (clusterin/sulfated glycoprotein ISGP-2], follicle-stimulating hormone [rFSH], alpha-inhibin, anti-Mullerian hormone, Wilms' tumor gene [WT-1], steroidogenic factor 1 [SF-1], SRY-related HMG box gene g [SOX9], and sex-determining region of Y chromosome) normally expressed in this cellular type. All were shown to express messenger ribonucleic acids for SGP-2, alpha-inhibin, WT-1, SOX9, and SF-1 (except SF-1 for clone no. 1). Moreover, we performed alkaline phosphatase and receptor tyrosine kinase p145 (c-kit) detection to ensure the absence of contamination by peritubular, germ cells, and Leydig cells. Both tests were negative for all the seven cell lines. These ovine Sertoli cell lines are the first ones obtained from livestock that exhibit specific Sertoli cell characteristics resembling different stages of phenotypic development. They provide useful in vitro model systems for toxicological investigations, coculture, and transfection experiments, making it possible to study signal transduction pathways, cell-cell interactions, and gene expression in species other than rodents.

Differentiated clonal cell lines were obtained from transgenic mice carrying a recombinant gene composed of DNA coding for a temperature-sensitive mutant of the simian virus large T antigen under the control of regulatory elements of the human vimentin gene. In response to mitogenic factors the vimentin promoter is activated in the presence of serum in almost all cultured cells independently of their origin. The expression of the T antigen could be controlled both at the level of transcription since the vimentin promoter is growth-regulated and at the level of the protein structure through the temperature stability of the T antigen. Indeed, the switch-off of the oncogene protein is obtained by serum deprivation of the culture and achieved with enhancement of the growth temperature. From transgenic mice several types of clonal differentiated cell lines were established and characterized including melanocytes, macrophages, mesangial, muscle, and endothelial cells. Melanocytes displayed melanin while endothelial cells from brain and heart expressed the related factor VIII and low density lipoprotein absorption capacities. Mesangial cells from kidney exhibited numerous desmosomes. Typical markers of macrophages from bone marrow were observed while skeletal muscle cells fused and contracted.

Gene expression in rabbit early development was investigated by microinjecting LacZ DNA and LacZ RNA in 1-cell and 2-cell embryos. Expression of LacZ DNA could not be obtained before 30–36 hpf, although synthetic LacZ RNA was translated from 12 hpf at the least. The onset of expression of microinjected DNA correlated with the 8- to 16-cell stage. This suggests that before this stage, there is a general negative control of gene expression. The arrest of in vitro development at the 2- to 8-cell stages did not inhibit LacZ expression, which still occurred at 33 hpf. In addition the inhibition of the first cleavage by nocodazole resulted in LacZ expression in 1-cell embryos. Expression of microinjected DNA thus occurs at a fixed time after fertilization and is independent of cleavages and of the second and subsequent DNA replications. Therefore, the changes in permissiveness for the expression of microinjected DNA in rabbit embryos are reminiscent of those in mouse embryos. Transcriptional selectivity in rabbit embryos was compared to that in early mouse embryos. In both species, Sp1-sensitive promoters were active and the promoter of simian virus 40 did not require far upstream enhancers before late cleavage stages; genes driven by the −447, +563 region of murine leukemia virus were repressed. In rabbit, however, the H-2K b promoter active in mouse was silent. Altogether, the results illustrate a remarkable conservation of the characteristics of the transcription in early rabbit and mouse embryos and the independence of its resumption from the pattern of cleavage.

The aim of this study was to investigate a new method to enhance the efficiency to create mammalian cell lines. Cell immortalization was achieved by intranuclear microinjection of a recombinant DNA construct composed of a constitutive promoter controlling the genes encoding immortalizing proteins; the sequences coding for the large T and small t antigens were fused downstream of regulatory elements from the vimentin gene, the activation of which characterizes the vast majority of cells growing in vitro. Data show that the efficiency of the immortalizing procedures using the SV40 early genes could be enhanced by the control elements derived from the human vimentin (HuVim) 5' sequences that contained nucleotides -878 to +93 from the CAP site. This HuVim 830-T/t recombinant was used to create cell lines from numerous primary cultures of different origins: rabbit, porcine and human endothelial cells, rabbit and bovine epithelial cells. A set of large T-expressing cells was derived, and these cells retained characteristics of differential cells: binding of Ulex europaeus lectin and synthesis of Factor VIII for human endothelial cells; network of cytokeratin for bovine oviductal cells and rabbit mammary cells.

The present paper describes a method of membrane preparation from ewe mammary gland using a sucrose cushion (1.3 M) to select smooth membranes; this results in a membrane preparation richer in PRL receptors than the microsomal preparation classically used. This method was used for the characterization and measurement of PRL and ovine placental lactogen (oPL) receptors in three organs of the ewe (mammary gland, liver, and adipose tissue). PRL receptors were measured by competition of iodinated human GH ([125I]hGH) with ovine PRL (oPRL). This hormone, which has both growth and lactogenic activities, appears to interact with PRL receptors with a higher affinity than oPRL itself and is a good probe for the determination of PRL receptors in the ewe. oPL receptors were measured by the specific binding of [125I]oPL. This hormone appears to bind exclusively to a somatogenic site in the ewe, since various GHs compete efficiently for binding, whereas oPRL is without effect. The evolution of PRL and oPL receptors was determined during pregnancy and lactation and at different periods after an estradiol and progesterone treatment, which provokes growth of the mammary gland and milk secretion. During pregnancy, PRL receptors increased in the mammary gland up to day 100. During the last trimester, receptor content remained stable, and a second increase occurred during early lactation. No additional significant changes were observed either for PRL receptors in liver or adipose tissue or for oPL receptors in any of the organs studied (mammary gland, liver, adipose tissue). Injections of large doses of estradiol and progesterone to nonpregnant ewes were able to reproduce effectively the pattern of PRL receptors observed during pregnancy, but had no effect on oPL receptor levels. These studies demonstrate the independence of PRL and PL receptor sites in the ewe and suggest a different hormonal regulation for each type of receptor.

The effects of progesterone on lactose synthesis activity and changes in mammary gland cells were studied in pseudopregnant rabbits simultaneously treated with prolactin. The injection of progesterone alone on Days 15 and 17 of pseudopregnancy decreased the activity of lactose synthetase (LSA) and galactosyl transferase (GTA), while the administration of prolactin for 2-4 days increased their activities. Th e simultaneous administration of progesterone and prolactin decreased the increase in LSA observed with prolactin alone by 70% on the 4th day of treatment, and decreased GTA by 30%. Progesterone completely suppressed the polyribosome profile and the ratio of endoplasmic reticulum bound polyribosomes to free polyribosomes induced by prolactin. The increase in RNA content in the mammary gland induced by prolactin was also suppressed by progesterone. The results suggest that progesterone inhibits the lactogenic action of prolactin without interfe ring with its mammogenic role.

SummaryProlactin receptors were maintained in organ cultures of rabbit mammary gland and this maintenance was related to an active synthesis since the addition of cyclo-heximide lead to a rapid decline in receptor levels. Inclusion of prolactin (5 μg/ml) in the culture medium resulted in an occupation of binding sites, followed by a reduction in the total receptor levels measured following dissociation of the bound prolactin from its receptor in crude membrane preparations. This apparent down-regulation of prolactin receptors may result from an endocytosis or compartmentalization of the hormone-receptor complex and may be involved in the mechanism of action of prolactin.

Prolactin injected into pseudopregnant rabbits led to a parallel enhancement of casein synthesis and casein mRNA concentration. When this stimulation was followed by a withdrawal of prolactin obtained by injections of bromocriptine, the rate of casein synthesis progressively diminished. In the presence of endogenous prolactin after the initial stimulation, the decline of casein synthesis was delayed. Hydrocortisone acetate injected with bromocriptine after the initial stimulation by prolactin was able to maintain a high rate of casein synthesis. Measurements of casein mRNA concentration by hybridization with casein cDNA indicated that in all cases the amount of casein mRNA was correlated with the magnitude of casein synthesis. This suggests that the lactogenic hormones, prolactin and glucocorticoids, which were previously demonstrated to be responsible for the enhancement of casein mRNA concentration are involved in their stabilization.

Pseudopregnant rabbit mammary glands in organ culture were used to investigate prolactin (PRL) receptor turnover. Chloroquine (100 microM) results in an increase in prolactin receptor levels (15.7 +/- 1.2% to 35.9 +/- 3.5% specific binding), whereas cycloheximide (1 microgram/ml) induces a rapid decline (to 6.4 +/- 1.2%) suggesting a rapid synthesis and degradation of the receptor molecule. Inhibitors of cellular transcription have little effect on receptor levels. Neither actinomycin D nor dichlororibofuranosylbenzimidazole (DRB) diminish PRL receptor levels whereas total protein synthesis is almost completely inhibited, and chloroquine increases the binding even in the presence of transcriptional inhibitors. These results imply that receptor synthesis continues and that the mRNA for the receptor protein is particularly stable. Ouabain (3 micrometers), which blocks the ATP-dependent Na+/K+ pump, provokes a greater than 60% reduction in PRL receptor levels without modifying total protein synthesis. Dinitrophenol (DNP, 1 mM), an oxidative uncoupler, has little effect on receptor levels, possibly due to a blockage of both synthesis and degradation. Prolactin is capable of inducing a 60% down-regulation of its own receptor, and this phenomenon appears to be energy-dependent because it is partially inhibited by DNP. This process seems to involve an increased rate of receptor degradation. These studies suggest that, at any one time, the level of PRL receptors in a target cell is the result of a dynamic equilibrium between receptor synthesis and degradation and that the most frequent modulations occur at the level of translation and lysosomal degradation. In conclusion, in mammary glands of the pseudopregnant rabbit, the prolactin receptor molecule appears to have a short half-life; the mRNA for this protein, however, is relatively stable.

Ovine placental lactogen is known to bind to prolactin receptors and to initiate milk synthesis in the rabbit mammary gland. However, this hormone exhibited a very low capacity of competing with 125 I-labeled human growth hormone for the binding to membranes extracted from ewe mammary gland. Ovine placental lactogen was very efficient in provoking the accumulation of β-casein mRNA in rabbit mammary explants but was much less active on ewe mammary explants. These data indicate that the placental hormone is not a potent lactogen in the homologous species and that its role in the control of mammary gland development and activity may have been previously overestimated.

Secretion of milk disappears very rapidly after removal of the pituitary gland in the ewe. The following modifications in mammary gland cells are associated with cessation of milk secretion: (1) a marked reduction in the number of secretory cells appears between 24 and 96 hr after hypophysectomy, as can be seen from the determination of the mammary gland DNA content and electron microscope examination; (2) an important decrease in the RNA content of the cells occurs before, and is greater than, the variation in the amount of DNA. It corresponds to a rapid inhibition of the 3 H-uridine incorporation into the RNA and results from the stimulation of RNA catabolism as measured by the increase in ribonuclease activity and the disappearance of the ribonuclease inhibitor. (3) there is a fall in the number of polyribosomes, particularly of those that are bound to the endoplasmic reticulum membranes, that is proportionately more pronounced than that of the total RNA. The polyribosomes maintain a synthetic activity that corresponds to their degree of aggregation.

Epithelial cells were isolated from mammary glands of pseudo-pregnant rabbits after a digestion of the tissue by collagenase and cell fractionation on a percoll gradient. These cells were cultured for various times in the presence of serum until growth was sufficient. The serum was then withdrawn for two days and hormones were added for one more day in the absence of serum. Cells were bound either directly to the plastic of the flask or to collagen spread in one or two layers. The concentration of β-casein mRNA was evaluated at the end of the culture by using a 3 H-DNA complementary to β-casein mRNA. The concentration of β3-casein mRNA was increased when prolactin was present in the culture medium. Cortisol amplified this effect while being totally inefficient alone. This induction was obtained as well when cells were cultured on plastic or on monolayer, bilayer or floating collagen, and also when they were kept in suspension. However, they apparently responded to prolactin only when fibroblasts were eliminated by adding hydroxy-L-proline (allo) or cholera toxin to the culture medium or by using collagen. The lactose synthetase activity and casein synthesis always remained extremely low regardless of the presence of hormones in the culture medium. Cells cultured on collagen tended to become reorganized in a way somewhat similar to that in mammary tissue, whereas cells spread on plastic remained uniformly distributed. These results suggest that isolated rabbit mammary epithelial cells have kept the essence of their capacity to respond to prolactin regardless of the culture technique. Isolated cultured cells thus appear to be a viable tool for further studies on the mechanism of prolactin action.

Spermidine concentration in rabbit mammary gland was estimated during pregnancy, lactation and after the induction of milk synthesis by prolactin and glucocorticoids in vivo and in vitro. It was observed that mammogenesis and lactogenesis during preganancy and the initiation of milk secretion at parturition are accompanied by an enhancement of spermidine concentration in the mammary gland. By contrast, the initiation of these phenomena by hormone injections does not require such variations of spermidine concentration. In organ culture, a slight increase in spermidine concentration was obtained under the influence of an hormonal combination including insulin, prolactin and cortisol. Spermidine added to the culture medium was unable to mimic cortisol action. An amplification of casein synthesis and a parallel increase of casein mRNA concentration was provoked by cortisol even when spermidine synthesis was blocked. Thus, one of the major actions of glucocorticoids during lactogenesis in the rabbit is not mediated through an increase in spermidine concentration in the mammary gland.

The onset of milk synthesis and secretion is the result of complex and multiple processes which operate during pregnancy and at parturition. Before pregnancy, the mammary gland is restricted to a few duct cells. During pregnancy, under the influence of estrogens, progesterone, and growth factors, the secretory cells progressively appear. They are organized in an epithelium forming a large number of alveoli. At the end of pregnancy, many alveolar cells are present and the development of the mammary gland is more or less complete according to species. After parturition, when milk secretion is triggered, the alveolar cells become polarized and hypertrophic. This transformation corresponds to the activation of the cells which have to elaborate and secrete huge amounts of proteins, lipids, and carbohydrates throughout lactation. Thus before being fully active, the mammary gland has been subjected to at least three types of transformation: (1) a cell multiplication which leads to the formation of alveoli, (2) an activation of specific genes directly involved in the elaboration of milk, and (3) an organization of the alveolar cells which become enriched in cellular organelles involved in the bulky production and secretion of milk. After weaning, the alveolar cells disappear until the next pregnancy.

The possibility of inducing maternal behaviour in non-pregnant ewes by injections of ovarian steroids has been investigated, using either a short course treatment (7 days) or a long term treatment (30 days). Short term treatment induced maternal behaviour with suckling in 14 out of 23 multiparous ewes and in 1 out of 13 nulliparous ewes while none of the control animals (7 multiparous and 4 nulliparous females) exhibited such a behaviour. The difference in the success of the treatment in multiparous and nulliparous females was significant. Long term treatment induced maternal behaviour in 9 out of 19 multiparous treated ewes while none of 11 ovariectomized control ewes displayed this behaviour and that 14 parturient ewes (out of 14) did. In both experiments there was a clear evidence of the establishment of a selective maternal behaviour shown by the treated ewes.

Milk synthesis is initiated solely by prolactin in the pseudopregnant rabbit and glucocorticoids potentiate this action of prolactin. In organ culture, prolactin, in the presence or in the absence of insulin, enhances casein synthesis and cortisol (inactive alone) amplifies this action. Measurements of casein mRNA concentration in total cellular RNA, by hybridization with DNA complementary to casein mRNA, revealed that the stimulation of casein synthesis by the glucocorticoid is accompanied by an increase in the amount of casein mRNA. A systematic comparison of variations of these two parameters indicated that the major effect of glucocorticoids on lactogenesis in the rabbit at this stage of mammary gland development is mediated through an increase in the quantity of casein mRNA available for translation. No simultaneous control of casein mRNA translation by cortisol was observed.

Resume La prolactine injectee pendant un jour a des lapines pseudogestantes induit l'accumulation des RNA messagers des caseines dans la glande mammaire. Trois jours apres la fin du traitement par la prolactine, les RNA messagers des caseines sont revenus a leur niveau de base chez les animaux traites par le CB 154, une drogue qui supprime la secretion de prolactine par l'hypophyse. Quand l'acetate d'hydrocortisone est injecte pendant les trois premiers jours en meme temps que le CB 154 apres le traitement initial par la prolactine, les RNA messagers des caseines se maintiennent a un haut niveau. Ce maintien n'a pas lieu lorsque la colchicine, qui est connue pour bloquer l'action inductrice de la prolactine, est injectee en meme temps que le steroide et le CB 154. En culture d'organes, la prolactine ajoutee au milieu de culture pendant seulement six heures est capable d'induire la synthese des caseines pendant au moins un jour apres avoir ete retiree du milieu de culture. Cette persistance de l'effet de la prolactine est supprimee par une addition d'anticorps anti-prolactine ou de colchicine au milieu de culture a partir de six heures. La prolactine ajoutee pendant seulement deux ou six heures a des milieux de culture de cellules epitheliales mammaires isolees, est capable d'induire l'accumulation des RNA messagers des caseines pendant au moins un jour apres que l'hormone ait ete retiree du milieu de culture. Cet effet de la prolactine est dans tous les cas supprime par l'addition d'anticorps anti-prolactine au milieu de culture. La colchicine ajoutee apres l'action initiale de la prolactine n'est inhibitrice que si la stimulation prolactinique etait d'une intensite moderee. Apres le retrait de la prolactine du milieu de culture, un nombre indecelable de recepteurs est occupe par l'hormone. Ces donnees suggerent qu'une quantite tres petite de prolactine peut se lier fermement a ses recepteurs. Cette association prolactine-recepteur est suffisante pour permettre la formation d'une quantite substantielle de relais intracellulaires de la prolactine portant l'information des recepteurs jusqu'aux genes des caseines. L'hormone qui reste fermement liee a ses recepteurs doit etre localisee essentiellement sur les membranes plasmiques puisque l'action de l'hormone peut etre arretee par des anticorps anti-prolactine. Apres un traitement aigu par la prolactine, une proportion notable des complexes prolactine-recepteur a ete transformee pour devenir refractaire a l'action de la colchicine.