A pig multi-tissue normalised cDNA library: large-scale sequencing, cluster analysis and 9K micro-array resource generation.

BACKGROUND: Domestic animal breeding and product quality improvement require the control of reproduction, nutrition, health and welfare in these animals. It is thus necessary to improve our knowledge of the major physiological functions and their interactions. This would be greatly enhanced by the availability of expressed gene sequences in the databases and by cDNA arrays allowing the transcriptome analysis of any function.The objective within the AGENAE French program was to initiate a high-throughput cDNA sequencing program of a 38-tissue normalised library and generate a diverse microarray for transcriptome analysis in pig species. RESULTS: We constructed a multi-tissue cDNA library, which was normalised and subtracted to reduce the redundancy of the clones. Expressed Sequence Tags were produced and 24449 high-quality sequences were released in EMBL database. The assembly of all the public ESTs (available through SIGENAE website) resulted in 40786 contigs and 54653 singletons. At least one Agenae sequence is present in 11969 contigs (12.5%) and in 9291 of the deeper-than-one-contigs (22.8%). Sequence analysis showed that both normalisation and subtraction processes were successful and that the initial tissue complexity was maintained in the final libraries. A 9K nylon cDNA microarray was produced and is available through CRB-GADIE. It will allow high sensitivity transcriptome analyses in pigs. CONCLUSION: In the present work, a pig multi-tissue cDNA library was constructed and a 9K cDNA microarray designed. It contributes to the Expressed Sequence Tags pig data, and offers a valuable tool for transcriptome analysis.

Longitudinal analysis of gene expression in porcine skeletal muscle after post-injection local injury.

PURPOSE: The purpose of this study is to describe the time course of gene expression in a skeletal muscle local injury induced by an intramuscular (IM) injection, and to compare the dynamics of gene expression with pathological events. MATERIALS AND METHODS: Ten piglets received 4 IM injections of propylene glycol in the longissimus dorsi muscles 6 h, 2, 7, and 21 days before euthanasia, where control and injected muscle sites were sampled for RNA isolation and microscopic examination. The hybridization of nylon cDNA microarrays was carried out with radioactive probes obtained from the muscle RNA. RESULTS: 153 genes were found under- or over-expressed at least once among the investigated time-conditions. The eight most discriminant genes were also identified: Two genes (GTP-binding protein RAD and Ankyrin repeat domain protein) were over-expressed at 6 h and six genes between 2 and 21 days (Osteonectin, Fibronectin, Matrix metalloproteinase-2, Collagen alpha 1(I) chain, Collagen alpha 2(I) chain, and Thymosin beta-4). Necrosis, inflammation and regeneration were observed through both the dynamics of gene expression profiles and through the microscopic examinations. CONCLUSION: Our data demonstrate that several pathways are involved in post-injection muscle injury, and that necrosis, inflammation and regeneration are not sequential but occur in parallel.

Expression of galectin-3 and its regulation in the testes.

Although spermatogenesis is a complex process under hormonal control, which includes mainly follicle stimulating hormone (FSH) and androgens, little is known about the intra-testicular mediators of these hormones. In the present study, galectin-3 (Gal-3) expression has been identified in human, rat and porcine testes where it is under hormonal control. Gal-3 is present in Sertoli cells and appears to be absent in human and (probably) in rat germ cells. Gal-3 expression was evidenced in the testes, in terms of both mRNA and protein (31 kDa). Gal-3 expression in cultured porcine Sertoli cells was shown to be under the positive control of FSH as well as of two cytokines epidermal growth factor (EGF) and tumour necrosis factor-alpha (TNF-alpha). Gal-3 expression in Sertoli cells is also potentially under the control of mature germ cells as an increased expression was observed in adult rat testes depleted in spermatocytes or spermatids. Although the function of testicular Gal-3 remains to be investigated, a potential role of Gal-3 in germ cell survival/regeneration is suggested based on its increased expression 1 month after a transient germ cell death process triggered by 10 days of treatment with the antiandrogen flutamide. Finally, although in the normal human testes, Gal-3 is exclusively located in the Sertoli cell cytoplasm, a nuclear localization is observed in the infertile testes. Together, the present findings have shown that (i) Gal-3 is expressed in the porcine, rat and human Sertoli cells; (ii) Gal-3 is under the positive control of FSH as well as of EGF and TNF-alpha and possibly of adult germ cells. These observations are compatible with a potential pro-survival role of Gal-3 in the testes.

Contribution to high-resolution mapping in pigs with 101 type I markers and progress in comparative map between humans and pigs.

In the frame of the European program GenetPig, we localized on the Pig map 105 coding sequences (type I markers) from different origins, using INRA-University of Minnesota porcine Radiation Hybrid Panel (IMpRH, 101 markers) and somatic cell hybrid panel (SCHP, 93 markers, of which only four were not also mapped using IMpRH). Thus, we contributed to the improvement of the porcine high-resolution map, and we complemented the integration between the RH and cytogenetic maps. IMpRH tools allowed us to map 101 new markers relatively to reference markers of the first generation radiation hybrid map. Ninety out of 101 markers are linked to an already mapped marker with a LOD score greater than 4.8. Seventy-eight markers were informative for comparative mapping. Comparison of marker positions on the RH map with those obtained on the cytogenetic map or those expected by Human-Pig comparative map data suggested to us to be cautious with markers linked with a LOD lower than 6. These results allowed us to specify chromosomal fragments well conserved between humans and pigs and also to suggest new correspondences (Sscr1-Hsap3, Sscr9-Hsap9, Sscr13-Hsap11, Sscr15-Hsap6) confirmed by FISH on pig chromosomes. We examined in more detail the comparative map between Hsap12 and Sscr5 considering gene order, which suggests that rearrangements have occurred within the conserved synteny.

The GENETPIG database: a tool for comparative mapping in pig (Sus scrofa).

The GENETPIG database has been established for storing and disseminating the results of the European project: 'GENETPIG: identification of genes controlling economic traits in pig'. The partners of this project have mapped about 630 porcine and human ESTs onto the pig genome. The database collects the mapping results and links them to other sources of mapping data; this includes pig maps as well as available comparative mapping information. Functional annotation of the mapped ESTs is also given when a significant similarity to cognate genes was established. The database is accessible for consultation via the Internet at http://www.infobiogen.fr/services/Genetpig/.

Glutathione S-transferase alpha expressed in porcine Sertoli cells is under the control of follicle-stimulating hormone and testosterone.

Glutathione S-transferases (GSTs) are a family of detoxification isoenzymes present in different tissues including the testis and that conjugate many toxic substrates to glutathione. Among these substrates are carcinogens, mutagens and products of oxidative processes. In the present report we show that GSTalpha is expressed in somatic testicular Leydig cells and Sertoli cells. GSTalpha expression in Sertoli cells is under the hormonal control of FSH, testosterone, and estradiol. In Leydig cells, immunoreactive GSTalpha was present at the neonatal, pubertal, and adult periods. In Sertoli cells, GSTalpha was predominant in pubertal and adult testes (but not in neonatal testes), suggesting that its expression is controlled by gonadotropins. The regulatory action and the mechanisms of action of FSH and testosterone on GSTalpha mRNA and protein levels were studied by using a model of primary cultures of porcine testicular Sertoli cells. FSH increased GSTalpha mRNA levels in a dose-dependent manner (ED50 = 18.5 nm/ml) with a maximal effect observed after 48 h of exposure (a 3-fold increase; P < 0.001). In addition, FSH increased GSTalpha protein, which was detected as a doublet of 28 kDa. Treatment with testosterone enhanced GSTalpha mRNA levels in a dose-dependent (ED50 = 1.4 ng/ml) and time-dependent manner with a maximal effect delayed at 8 h of exposure (a 2-fold increase; P < 0.001). Similarly, Sertoli cell treatment with testosterone metabolites, dihydrotestosterone (DHT) and estradiol, led to an increase in GSTalpha mRNA levels. Because stimulatory effects of FSH and androgens were also observed on GSTalpha protein, we therefore had to determine whether the different hormones were affecting GSTalpha gene transcriptional activity, or GSTalpha mRNA stability, or both. FSH and 8-Br-cAMP (but not testosterone) increased the stability of GSTalpha mRNA. The effects of FSH and testosterone on GSTalpha protein were additive, confirming that both hormones act through distinct mechanisms on the expression of the enzyme. Taken together, the present observations indicate that Sertoli cell GSTalpha is targeted by FSH, testosterone, and its metabolites, and they reinforce the concept that Sertoli cells exert a protective role and are under endocrine control to ward against toxic agents in the context of Sertoli-germ cell interactions during spermatogenesis.