Fine mapping of the FecL locus influencing prolificacy in Lacaune sheep.

In the Lacaune sheep population, two major loci influencing ovulation rate are segregating: FecX and FecL. The FecX(L) mutation is a non-conservative substitution (p.Cys53Tyr) in BMP15 that prevents the processing of the protein. Using a statistical approach, FecL has been shown to be an autosomal major gene. A full genome scan localized the FecL locus on sheep chromosome 11. Fine mapping reduced the interval containing FecL to markers BM17132 and FAM117A, corresponding to a synteny block of 1.1 megabases on human chromosome 17, which encompasses 20 genes. The expression of 16 genes from this interval was observed in tissues of the reproductive axis, but expression was not affected in homozygous FecL(L) females. In this interval, a unique haplotype was associated with the FecL(L) mutation. This particular haplotype could be predicted by the DLX3:c.*803A>G SNP in the 3' UTR sequence of the DLX3 gene. This SNP provided accurate classification of animals (99.5%) as carriers or non-carriers of the mutation and therefore maybe useful in marker assisted selection. A synergistic action of FecL(L) and FecX(L) mutations on both ovulation rate and litter size was demonstrated. Until now, all the Fec genes identified in sheep belong to the bone morphogenetic protein (BMP) system. Based on the human orthologous region, none of the 20 genes in the FecL region corresponds to known molecules in the BMP system. The identification of the FecL(L) mutation could lead to the discovery of a new pathway involved in the regulation of ovulation rate.

A deletion in the bone morphogenetic protein 15 gene causes sterility and increased prolificacy in Rasa Aragonesa sheep.

Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor beta superfamily, is specifically expressed in oocytes and is essential for sheep prolificacy. Reported mutations in this gene cause increased ovulation rate and infertility in a dosage-sensitive manner. In this work, a new naturally occurring mutation in the BMP15 gene from the ovine Rasa Aragonesa breed is described. This mutation is a deletion of 17 bp that leads to an altered amino acid sequence and introduces a premature stop codon in the protein. Highly significant associations (P < 0.0001) were found between the estimated breeding value for prolificacy and the genotype of BMP15 in Rasa Aragonesa animals with high and low breeding values for this trait. As for other mutations in BMP15, this new mutation is associated with increased prolificacy and sterility in heterozygous and homozygous ewes respectively.

A novel method to isolate the common fraction of two DNA samples: hybrid specific amplification (HSA).

Hybrid specific amplification (HSA) is a novel simple method elaborated in order to isolate the common fraction of two DNA samples while avoiding the background due to repeated sequences. The method is based on the suppressive PCR principle, associated with a Cot1 pre-hybridization step. In recent work we demonstrated that hyperprolificity observed in Booroola ewes is associated with a mutation in the bone morphogenetic protein receptor IB gene (BMPR-IB). We applied HSA between ovarian cDNA and DNA from four BAC clones containing BMPR-IB in order to test for the presence of other genes expressed in ovary and to isolate additional BMPR-IB exon sequences. Of the 460 clones obtained, none contained repeated sequences. We successfully obtained 37 clones representing the major part of BMPR-IB coding sequence, together with 5'- and 3'-UTR sequences. Here we have successfully applied HSA to a particular tissue, but it should be possible to trap the common fraction of two DNA samples, whatever their nature.

Using markers in gene introgression breeding programs.

We investigate the use of markers to hasten the recovery of the recipient genome during an introgression breeding program. The effects of time and intensity of selection, population size, number and position of selected markers are studied for chromosomes either carrying or not carrying the introgressed gene. We show that marker assisted selection may lead to a gain in time of about two generations, an efficiency below previous theoretical predictions. Markers are most useful when their map position is known. In the early generations, it is shown that increasing the number of markers over three per non-carrier chromosome is not efficient, that the segment surrounding the introgressed gene is better controlled by rather distant markers unless high selection intensity can be applied, and that selection on this segment first can reduce the selection intensity available for selection on non-carrier chromosomes. These results are used to propose an optimal strategy for selection on the whole genome, making the most of available material and conditions (e.g., population size and fertility, genetic map).

The Booroola mutation in sheep is associated with an alteration of the bone morphogenetic protein receptor-IB functionality.

The hyperprolificacy phenotype of Booroola ewes is due to the presence of the FecB(B) allele at the FecB locus, recently identified as a single amino acid substitution (Q249R) in the bone morphogenetic protein (BMP) type-IB receptor (BMPR1B), and is associated with a more precocious differentiation of ovarian granulosa cells (GCs). To evaluate the consequences of the Booroola mutation on BMPR1B functions, the action of ligands of the transforming growth factor-beta (TGFbeta)/BMP family that act through (growth and differentiation factor-5, BMP-4) or independently of (activin A, TGFbeta-1) BMPR1B were studied on primary cultures of GCs from homozygous FecB(+) and FecB(B) ewes. All the tested TGFbeta/BMP family ligands inhibited progesterone secretion by FecB(+) GCs. Those inhibitory effects were lower for GCs from preovulatory (5-7 mm diameter) than from small antral follicles (1-3 mm diameter). The presence of the Booroola mutation was associated with a 3- to 4-fold (P<0.001) decreased responsiveness of GCs from FecB(B) compared with FecB(+) small follicles to the action of BMPR1B ligands. In contrast, TGFbeta-1 and activin A had similar inhibitory effects on progesterone secretion by GCs from FecB(+) and FecB(B) small follicles. No difference between genotypes was observed with GCs from preovulatory follicles. In transfection experiments with HEK-293 cells, co-expression of FecB(+) BMPR1B and BMPR2 resulted in a 2.6-fold (P<0.01) induction of the activity of a BMP-specific luciferase reporter construct by BMP-4. Interestingly, no response to BMP-4 was observed when cells were transfected with the FecB(B) form of the BMPR1B receptor. Overall, these data strongly suggest that the Q249R mutation is associated with a specific alteration of BMPR1B signaling in hyperprolific Booroola ewes.

Protein kinase C inhibition of in vitro FSH-induced differentiation in pig granulosa cells.

In granulosa cells, growth factor IGF I plays a major role in both growth and differentiation, acting through an autocrine/paracrine mechanism, and its production is regulated by FSH, via cyclic AMP (cAMP). As protein kinase C is also involved in granulosa cell function, we investigated the possibility that its activation could balance the positive effects of FSH. Using pig granulosa cells cultured in vitro, we studied the effects of protein kinase C activation by tetradecanoylphorbol acetate (TPA) on IGF I mRNA level. We also checked morphological modifications, cAMP production and steroidogenesis at the P450 side chain cleavage mRNA and progesterone levels. Our data demonstrate that protein kinase C activation antagonizes the in vitro FSH-induced differentiation, particularly morphological modifications and accumulation of IGF I mRNA. These inhibitory effects on FSH responses suggest that there could be a balance between protein kinase A and protein kinase C pathways in regulating differentiation in pig granulosa cells.

Gonadotropins induce accumulation of insulin-like growth factor I mRNA in pig granulosa cells in vitro.

Pig granulosa cells have been shown to synthesize insulin-like growth factor (IGF) I peptide in vitro, and this expression is regulated by gonadotropins via the cAMP pathway. By hybridizing an IGF I cDNA probe with total RNA isolated from pig granulosa cells cultured in vitro, we show that these cells contain two IGF I transcripts of about 0.9 kb and 9 kb in size. Treatment of the cells with gonadotropins (follicle-stimulating hormone, luteinizing hormone) or cAMP agonists (dibutyryl-cAMP, forskolin) induces an accumulation of the transcripts which can be abolished by transcriptional inhibitors, but not by translational inhibitors. We thus provide new evidence that pig granulosa cells are a site of IGF I synthesis, and we conclude that (1) gonadotropins increase IGF I mRNA levels; (2) the accumulation of IGF I mRNA results from an increased transcription; (3) the stimulation of IGF I gene transcription does not require ongoing protein synthesis; (4) these effects of follicle-stimulating hormone can be mimicked by cAMP agonists.

[Long-term outcome of 72 patients surgically treated for stage II non-small cell bronchial cancer, between 1982 and 1989]. / Devenir à long terme de 72 patients opérés d'un cancer bronchique non à petites cellules, de stade II, entre 1982 et 1989.

We report a 72 patients trial, who had surgical treatments for non small cell lung cancer, stage II (T1N1, T2N1). In our retrospective study, the overall 5-years survival is 44%, with a 24-month median survival. 45% of the patients have recurrence mainly due to distant metastasis. State II appears to be an heterogeneous group. As shown in other studies, the presence of hilar nodes (N1H) seems to be linked with a pejorative outcome. In our series, the survival associated with Lobar N1 (N1L) disease is the same as the survival of Hilar N1 disease, but the initial sites of recurrence differ. The interest of a postchirurgical treatment is controversial. The postoperative radiotherapy reduces the local recurrence without increasing the survival. The chemotherapy treatment is debatable and several studies are under way. We reviewed the different causes of death. The appearance of second cancer in the cured patients is very frequent.

[Spontaneous hydro-pneumothorax disclosing malignant mesothelioma. Apropos of 2 cases]. / Hydro-pneumothorax spontané révélant un mésothéliome malin. A propos de deux cas.

We report the cases of two males who presented with spontaneous complete unilateral pneumothorax with ipisilateral liquid effusion. Neither had a history of previous respiratory disease. In both cases chest tube drainage resulted in recurrence of pneumothorax with chronic illness requiring surgical exploration. The surgery revealed a malignant pleural mesothelioma by histological examination. Thus, spontaneous pneumothorax, particularly with abondant effusion can be a revealing symptom of malignant pleural mesothelioma.

[Endoscopic management of broncho-pleural fistula in a patient with acute respiratory distress syndrome after pneumonectomy]. / Prise en charge endoscopique d'une fistule bronchopleurale chez un patient en syndrome de détresse respiratoire aiguë après pneumonectomie.

We report the management of endobronchial a patient admitted to the ICU for respiratory distress in the consequences of an surgical recovery of his left pneumonectomy complicated by bronchopleural fistula as part of a bronchial carcinoma non-small cell type adenocarcinoma. Endobronchial treatment by gluing of the fistula may be an alternative to surgery. We discuss its indication in the treatment of bronchial fistula.

Selection and preliminary characterization of cycloleucine-resistant CHO cells affected in methionine metabolism.

Cycloleucine is in vivo a potent inhibitor of S-adenosylmethionine (SAM) biosynthesis and subsequent methylation reactions in somatic mammalian cells. Cycloleucine-resistant (CLr) clones were isolated from CHO cells by single-step selection. Their phenotype was stable when they were grown in the absence of drug. These clones appeared randomly in cultures at the frequency of 5 x 10(-6)/cell/generation, as determined by a fluctuation test. EMS mutagenesis did not significantly increase this frequency. The cycloleucine-resistant phenotype was codominant in intraspecific hybrids. Cycloleucine-resistant clones showed increased SAM pools; on the contrary, methionine pools were not significantly affected in these clones when compared to the wild-type cells. This increased SAM production was correlated with an increase of methionine adenosyltransferase (MAT) SPECIFIC ACTIVITY IN RESISTANT CLONES UNDER VARIOUS GROWm. The mechanism of posttranscriptional control of MAT biosynthesis was not affected in the cycloleucine-resistant clones nor were the kinetic properties of the enzyme modified. The genetic or epigenetic origin of this resistance mechanism is discussed.

Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium.

A new synthetic medium (referred to as GC3) that supports the growth of the Chinese hamster ovary cell line has been developed. It is composed of a 1:1 mixture of Ham's F12 and modified Eagle's minimum essential (MEM.S) mediums supplemented with transferrin (10 micrograms/ml), insulin (80 mU/ml), and selenium (1 X 10(-7) M). Other more simple supplementations of our basal medium MEM.S/F12 (transferrin + insulin, transferrin + selenium, ferrous iron + selenium) also give good cell growth responses. Fibronectin or serum pretreatment is not needed for cellular attachment and spreading. Our culture system is characterized by a continuous serum-free cultivation (more than 200 doublings), a clonal growth, a high density proliferation, and a rapid growth rate near that of cells in serum-supplemented medium.

Phleomycin resistance as a dominant selectable marker in CHO cells.

The Tn5 and the Streptoalloteichus hindustanus (Sh) ble genes conferring resistance to bleomycin-phleomycin antibiotics have been cloned into a mammalian vector under the RSV-LTR promoter. The resulting plasmids, pUT506 and pUT507 respectively, were used to transfect CHO cells by either the calcium phosphate or the recently described polybrene-DMSO method. Phleomycin- or bleomycin-resistant clones arose with a higher frequency after transfection with pUT507, and pUT507 transfectants were more resistant to both antibiotics than pUT506 transfectants. Phleomycin resistance in pUT507 transfectants was stable and associated with integration of plasmid sequences in genomic DNA. The Sh ble gene, which confers a dominant phleomycin-resistance phenotype, should provide a useful transferable selectable marker in CHO cells as well as in other animal cell lines.

Denaturing gradient gel electrophoresis detection of polymorphism in a PCR fragment of sheep epidermal growth factor gene.

The ovine map is not yet well-developed, which represents a problem when looking for markers of a region of interest in sheep. A means of circumventing this is to use comparative mapping. In this study primers were determined using consensus sequences for the epidermal growth factor gene of humans, rats and mice, and an ovine epidermal growth factor gene fragment was amplified by polymerase chain reaction (PCR). A new set of specific ovine primers was chosen to study the polymorphism of this DNA fragment by denaturing gradient gel electrophoresis. Eighty-four individuals belonging to seven sheep breeds were studied with this technique and four alleles were detected. The heterozygosity rate was 0.57. Family analysis showed mendelian inheritance of the alleles. Usually, genetic analysis of type-I loci used in the comparative mapping is based on the detection of restriction fragment length polymorphisms in sheep DNA using cDNA probes from other species. Our work shows that another method, based on PCR and denaturing gradient gel electrophoresis techniques, can be efficiently used.

Regional localisations of VIM, HSD3b, ACTA1 and PGM1 in pigs.

The comparative map between human and pig has progressed rapidly over the past 2 years. Nevertheless, some points still need to be clarified, particularly the correspondences between human chromosome 10 (HSA10) and porcine chromosome 10 (SSC10) and between human chromosome 1 (HSA1) and porcine chromosomes. The gene codings for vimentin (VIM) carried by HSA10 and three genes carried by HSA1 (hydroxy delta 5 steroid dehydrogenase 3 beta: HSD3B: alpha actin 1: ACTA1: and phosphoglucomutase 1: PGM1) were selected and the regional localisations on pig chromosomes were determined using a well-characterised somatic cell hybrid panel.

P450scc regulation in pig granulosa cells: investigation into the mechanism of induction.

P450scc catalyses the first and rate-limiting reaction in steroidogenesis and is hormonally regulated. By Northern analysis, using a bovine cDNA probe, we have studied the regulation of P450scc mRNA in pig granulosa cells cultivated in vitro. Using transcription and translation inhibitors, we show that the gonadotropin-induced accumulation of P450scc mRNA mainly results from increased transcription, and that this stimulation, at least in part, is protein synthesis-dependent. Although transcriptional regulation of P450scc gene expression is found in other steroidogenic cells, cycloheximide-sensitivity of this regulation is not widespread. Pig granulosa cells thus would constitute a useful model to study this mechanism of regulation.

Different levels of human intervention in domestic rabbits: effects on genetic diversity.

The effects of human interaction on domestic rabbits were evaluated through the analysis of animals (up to 267) belonging to fancy breeds (22), a commercial breed (1), and selected strains (2). Microsatellite loci and mtDNA polymorphism revealed that the genetic pool of domestic rabbits studied only originated from that available in France. The good conservation of the original diversity was probably ensured through the multiplicity of samplings from wild populations. Selected strains, because of the breeding strategy, keep a fairly high level of diversity compared to other breeds.

Synteny conservation between parts of human chromosome 4q and bovine and ovine chromosomes 6.

Booroola Merino sheep are characterized by a high ovulation rate attributable to the presence of the FecB allele of the FEC gene. This gene, which has been assigned to sheep chromosome 6, is linked to the gene for EGF, which in man is located on the long arm of chromosome 4 (HSA 4q). To increase the number of known markers on sheep chromosome 6, we used comparative mapping data from sheep, man, and cattle. In our study, we show a synteny between EGF and the genes PDHA2 and FGF5 (from HSA 4q) and microsatellites ILSTS018, ILSTS090, and ILSTS093 (from bovine chromosome 6) in sheep. We also show that the conservation between HSA 4q and sheep chromosome 6 is disrupted between EGF and FGF2.

A first catalog of genes involved in pig ovarian follicular differentiation.

As a first step toward the characterization of genetic expression in pig ovaries, we have selected 238 clones by differential hybridization from a pig granulosa cell cDNA library, using probes prepared from RNA extracted from either untreated or FSH-treated cells and, in order to generate expressed sequence tags (ESTs), we have performed 3' and 5' single-pass sequencing of these clones. Sequences of the 3' end of the 167 clones that produced informative sequence data were first compared with each other, revealing a redundancy level of 21%. Sequences from the 136 unique clones were analyzed for similarities with sequence data included in Genbank and EMBL databases. Among these unique clones, 54 (40%) matched significantly with sequences from either Genbank of EMBL: 4 with known genes in pig, 35 matched with previously reported human genes, and 15 with other mammalian genes. Eighty-two clones (60%) showed no significant match with any gene or DNA sequence in the Genbank and EMBL databases and thus may represent new pig transcripts.