Claude Delouis

Role of prolactin and glucocorticoids in the expression of casein genes in rabbit mammary gland organ culture. Quantification of casein mRNA

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.

Mammalian cell lines can be efficiently established in vitro upon expression of the SV40 large T antigen driven by a promoter sequence derived from the human vimentin gene

Summary— 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 differentiated 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.

Stabilization of casein mRNA by prolactin and glucocorticoids

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.

Prolactin receptor turnover in explants of pseudopregnant rabbit mammary gland.

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.

Comparative measurement of the lactogenic activity of ovine placental lactogen in rabbit and ewe mammary gland

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 125I-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 beta-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.

Prolactin receptors in organ culture of rabbit mammary gland: effect of cycloheximide and prolactin.

Summary Prolactin 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.

Role of spermidine in casein gene expression in the rabbit

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.

Transcription of Y‐ and X‐linked genes in preimplantation ovine embryos

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.

Expression of microinjected DNA and RNA in early rabbit embryos: Changes in permissiveness for expression and transcriptional selectivity

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-2Kb 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.

ESTABLISHMENT AND CHARACTERIZATION OF IMMORTALIZED OVINE SERTOLI CELL LINES

SummaryThe 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 [SGP-2], follicle-stimulating hormone [rFSH], α-inhibin, and sex-determining region of Y chromosome) normally expressed in this cellular type. All were shown to express messenger ribonucleic acids for SGP-2, α-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.