In the sheep as in many mammalian species, growth and atresia of antral follicles are characterized, respectively, by a decrease and a high increase in the intrafollicular levels of insulin-like growth factor binding proteins of less than 40 kDa (IGFBPs < 40 kDa), mainly IGFBP-2, -4, and -5. The objective of this study was to investigate whether such changes are associated with changes in follicular expression of the corresponding mRNA. For this purpose, ovaries were recovered from ewes slaughtered at the end of follicular phase (i.e., 30 h after progestagen sponge removal; control ewes) or at 24 h, 36 h or 72 h after hypophysectomy (hypox) performed 30 h after sponge removal. The expression of mRNA of IGFBPs of less than 40 kDa (IGFBPs < 40 kDa mRNA) was studied in ovine antral follicles from control and hypox ewes by in situ hybridization using [35S]-labeled human IGFBP-2, -4, and -5 cRNA as probes. In control ewes, IGFBP-2 mRNA was mainly expressed in granulosa as a gradient in healthy follicles, the expression being higher in granulosa cells close to the basal membrane than in granulosa cells bordering the antrum and within the cumulus. The level of IGFBP-2 mRNA was lower both in granulosa cells close to the basal membrane and in those bordering the antrum from small follicles than in the corresponding compartments of granulosa cells from large healthy follicles (p < 0.05). In healthy follicles, IGFBP-4 and -5 mRNA were mainly expressed in thecal cells. No change in level of IGFBP-4 mRNA was observed between small and large follicles, whereas the level of IGFBP-5 mRNA tended to be lower in thecal cells from large compared to small follicles (p = 0.055). In atretic follicles, expression of IGFBPs < 40 kDa mRNA strongly increased in granulosa (IGFBP-2 and -5, p < 0.01) and in thecal cells (IGFBP-2 and -4, p < 0.01). In hypox ewes, the chronology of changes in expression of follicular IGFBPs < 40 kDa mRNA and in intrafollicular levels of the corresponding proteins was studied during atresia of large antral follicles. Early atresia of large follicles was associated with a strong decrease in intrafollicular estradiol levels (p < 0.001); an increase in intrafollicular levels of IGFBP-2, -4, and -5 (p < 0.001) an increase in both IGFBP-2 (p < 0.001) and -5 (p < 0.01) mRNA expression in granulosa and thecal cells; but no changed in IGFBP-4 mRNA expression. Late atresia of large follicles was associated with a further decrease in intrafollicular estradiol levels (p < 0.01); a further increase in intrafollicular levels of IGFBP-2, -4, and -5 (p < 0.001); an increase in IGFBP-4 (p < 0.01) and -5 (p < 0.05) mRNA expression in theca and granulosa, respectively; a decrease in IGFBP-5 mRNA expression in theca (p < 0.05); but no further increase in IGFBP-2 mRNA expression. Overall, these data suggest that the decrease and the increase in expression of mRNA of follicular IGFBPs < 40 kDa during follicular growth and atresia, respectively, are involved in the decrease and the increase in intrafollicular levels of the correspond...

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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.

Expressed sequence tags (ESTs) are partial sequences from the extremities of complementary DNA ( C DNA) resulting from a single pass sequencing of clones from cDNA libraries, and different ESTs can be obtained from one gene. Sequence information from ESTs can be used for deciphering the function and the organisation of the genome. From a functional viewpoint, they allow the determination of the expression profiles of genes in any particular tissue, in different conditions or status, and thus the identification of regulated genes. In order to identify genes involved in particular processes one can select a specific group of mRNAs. For such a selection, classical techniques include subtraction or differential screening and new techniques, using polymerase chain reaction (PCR) amplification, are now available. For studies on the organisation of the genome the main use of ESTs is the determination of chromosomal localisation of the corresponding genes using a somatic hybrid cell panel. This chromosomal localisation information is needed to identify genes or quantitative trait loci, according to the 'positional candidate' approach. ESTs also contribute to comparative genetics and they can help to decipher gene function by comparison between species, even genetically distant ones. Thus, combining sequence, functional and localisation data, ESTs contribute to an integrated approach to the genome.Résumé -Des étiquettes pour les gènes : une revue. Les « étiquettes » correspondent aux séquences des extrémités des ADN complémentaires, obtenues de manière systématique à partir d'une seule réaction de séquençage. Cependant, à partir d'un

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