Pierre Gaye

Cellular localization of an embryonic interferon, ovine trophoblastin and its mRNA in sheep embryos during early pregnancy

Summary— The ovine embryo produces an interferon named ovine Trophoblastin (oTP) which is involved in the maternal recognition of pregnancy and ensures the maintenance of progesterone secretion by the corpus luteum. We have used indirect immunohistofluorescence and in situ hybridization on histological sections to investigate the fate of this protein and its mRNA in ovine embryos from days 3 to 25 of pregnancy. The level of expression was measured by image analysis of the autoradiographs after in situ hybridization. Both techniques clearly demonstrated that oTP and its mRNA were specifically localized in the extra‐embryonic trophoblast. Neither the embryonic cells, nor the yolk sac or the amniotic tissues produced the protein or its mRNA. The protein could be detected by d 11 of pregnancy in the elongated blastocyst. Maximum of expression is observed at d 14 and the level decreased by d 16 of pregnancy. The arrest of expression occurred in the regions of trophoblast which have established cellular contacts with the uterine epithelium during the implantation process.

Cloning and expression of cDNA encoding ovine trophoblastin: its identity with a class-II alpha interferon

The cDNAs encoding ovine trophoblastin (oTP) were isolated from an ovine embryo cDNA lambda gt 11 library by screening with a synthetic 29-mer oligodeoxynucleotide corresponding to amino acid (aa) residues 34 to 43 of oTP. The cDNA contained an open reading frame of 595 bp and the deduced amino acid sequence indicates a protein precursor of 195 aa. Nucleotide and amino acid sequence comparisons establish that oTP shares extensive homology with alpha-interferon (IFN-alpha) but is more closely related to the IFN-alpha sII subfamily. When the oTP cDNA was cloned into an eukaryotic expression vector and transfected in monkey COS cells, a high level of antiviral activity was detected. RNA blot analyses of total RNA reveal that the oTP-coding gene is expressed during a relatively short period (eleven to 21 days). The abundant expression of oTP mRNA corresponds closely to the time at which the embryo acts to extend luteal lifespan. RNAs homologous to oTP were also detected in goat and cow embryos at equivalent periods of their development, but not in the pig.

Regulation of casein synthesis in the rabbit mammary gland: Titration of mRNA activity for casein under prolactin and progesterone treatments

Total polysomal RNA or poly(A)-containing RNA isolated from membrane-bound polysomes of normal lactating rabbits directed the synthesis of casein in a reticulocyte lysate. Casein was identified by immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis of the immunoprecipitate. The poly(A)-containing RNA was heterogeneous with one major peak corresponding to a 12-S sedimentation coefficient as determined by polyacrylamide gel electrophoresis. Using the same procedure, mRNAs isolated from the non-secreting tissue of pseudopregnant rabbits were found not to contain the 12-S peak and were unable to direct the synthesis of casein in vitro. Prolactin injected into pseudopregnant rabbits induced the synthesis of proteins immunoprecipitable by anti-casein anti-serum and induced the simultaneous appearance of the 12-S mRNA. Progesterone injected with prolactin prevented the induction of casein synthesis and the appearance of mRNA for casein. A close relationship was established between the ability of the tissue to synthesize immunoprecipitable casein and the corresponding mRNA content of polysomes.

Complete nucleotide sequence of bovine α-lactalbumin gene: comparison with its rat counterpart

Abstract The nucleotide sequence of the bovine α-lactalbumin gene, whose organization is very similar to that of its rat counterpart, was deduced from the analysis of 2 λ clones isolated from a HindIII genomic bank. The 3090 sequenced nucleotides comprise 738 bp upstream from the transcription unit (∼2 kb) which contains 4 exons of 160, 159, 76 and 330 bp separated by 3 introns of 321, 473 and 504 bp. Comparison with the rat α-lactalbumin gene shows similar percentages of homology between the 4 cognate exons. Since only the first three exons are homologous to the corresponding exons of the lysozyme gene, it is suggested that the 4th exons of α-lactalbumin and lysozyme genes have different origins. The bovine α-lactalbumin mRNA is 725 nucleotides long, excluding the poly(A) tail. The reading frame and the flanking 5′ and 3′ untranslated regions contain 429, 27 and 269 nucleotides, respectively. The derived amino acid sequence differs at 10 positions from that determined directly on mature α-lactalbumin.

Identification and characterization of growth hormone receptor mRNA in the mammary gland.

The present report describes the first characterization of growth hormone (GH) receptor (GH-R) mRNA in the rabbit mammary gland. Northern blot analysis of poly(A)+ RNA isolated from several tissues of rabbit probed with a rabbit liver GH-R cDNA fragment revealed hybridization to only one transcript of 4.2 kb. A specific hybridizing signal appears in the mammary gland mRNA during gestation, when three different probes derived from liver GH-R cDNA and encoding respectively for extracellular, transmembrane and intracellular regions, were used. The signal is lower than in the liver but highly significant. These results indicate that the three regions are present and well conserved in the GH-R transcript found in the mammary gland. By S1 nuclease mapping analysis we demonstrated that the extracellular and transmembrane domains of mammary gland GH-R mRNA are strongly homologous to the liver GH-R mRNA. In addition, mammary gland GH-R mRNA is probably generated by mammary epithelial cells as demonstrated by the hybridization signal obtained using mRNA extracted from purified acini. The increase in the concentration of GH-R mRNA occurs during epithelial cell proliferation associated with a decrease in the proportion of adipocytes and connective cells at late gestation. The 4.2 kb GH-R mRNA species was also detected in ovine and porcine mammary glands during gestation, suggesting a probable expression of the related form of GH-R in these species.

Amino terminal sequences of the precursors of ovine caseins

Abstract The amino terminal sequences of the 4 caseins synthesized by translation of ovine mammary mRNAs in a wheat germ cell-free system have been investigated by automated Edman degradation. The 3 “calcium-sensitive” caseins ( α s1 , α s2 and β) and κ-casein were synthesized as precaseins with amino terminal hydrophobic extensions of 15 and 21 amino acid residues respectively, resembling “signal peptides” of other secretory proteins. The extra pieces of the 4 caseins, which start with a methionyl residue, end with an alanyl residue which may be one of the signals recognized by the mammary membrane-bound enzyme responsible for the specific cleavage of precaseins. The amino terminal extensions of α s1 , α s2 and β-caseins show a high degree of homology suggesting that they have derived from a common ancestor.

Preferential synthesis of β lactoglobulin by the bound polyribosomes of the mammary gland

Abstract The free polyribosomes of the mammary gland of the ewe in lactation, in spite of being very active in incorporating amino-acids into proteins, nevertheless have potentialities different from those of the bound polyribosomes for the elaboration in vitro of β lactoglobulin. The latter protein is almost exclusively synthetized by the forms bound to the membranes of the endoplasmic reticulum.

Cloning and structural analysis of two distinct families of ovine interferon-α genes encoding functional class II and trophoblast (oTP) α-inteferons

Abstract Ovine trophoblast protein (oTP) is a polypeptide secreted by ovine trophectoderm from day 11 to 21, which plays a key role in maternal recognition of pregnancy. Structural analyses established that oTP shares extensive homology with class II α-interferon (IFN-αII) subfamily. Previous screening of an ovine genomic DNA library probed with an oTP cDNA incidently resulted in the isolation of a functional IFN-αII gene and two relevant pseudogenes, as shown by sequence analysis and study of expression in eukaryotic COS cells. The expected oTP gene together with a cognate pseudogene was successfully isolated from the series of clones selected from another genomic library probed with the oTP cDNA, using two specific oligonucleotides, each one complementary to a region of oTP cDNA with little homology with the IFN-αII gene and related pseudogenes. Southern blotting of ovine genomic DNA indicated the existence of at least five trophoblast IFN-α genes or pseudogenes. Nucleotide sequence comparisons showed that the oTP gene exhibits a higher homology (90%) with bovine trophoblast IFN gene (Stewart et al. (1990) J. Mol. Endocrinol. 4, 275–282) than with oIFN-αII gene (70%), thus providing evidence that embryonic IFNs constitute a distinct subfamily of IFN-αs.

Amino terminal sequence of the precursor of ovine β-lactoglobulin

Abstract Ovine mammary gland mRNAs were translated in a wheat-germ cell-free system in the presence of radioactive amino acids. Automated Edman degradation performed on α-lactalbumin isolated by immunoprecipitation from the mixture of radiolabelled lactoproteins showed the occurrence of an hydrophobic 19 residues long amino terminal extension. The pre-protein represents the primary translation product since the amino terminal methionyl residue was found to be donated by initiator Met-tRNA i Met . Comparison of the signals of ovine α-lactalbumin and hens egg white lysozyme, two homologous proteins which are thought to be derived from a common ancestor, suggests that the signal region has evolved at least as rapidly as the remaining part of the polypeptide chain.

The bovine α-lactalbumin promoter directs expression of ovine trophoblast interferon in the mammary gland of transgenic mice

A hybrid construct derived from ovine trophoblastin cDNA and bovine α‐lactalbumin‐encoding gene, was injected into the pronuclei of mouse eggs. In one of the resulting transgenic mouse lines, expression of the hybrid construct was detected and found to be limited to the mammary gland of lactating females which secreted active ovine trophoblastin. This strongly suggests that important cis‐acting DNA sequences involved in tissue‐specific expression of the bovine gene are located within the second half of the 3′ untranslated region, or/and the proximal 5′and 3′ regions flanking the transcriptional unit.