François Gasser

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

SummaryA 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 Hams F12 and modified Eagles minimum essential (MEM.S) mediums supplemented with transferrin (10 μg/ml), insulin (80 mU/ml), and selenium (1×10−7M). 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.

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.

A genetic study of various enzyme polymorphisms in Pleurodeles waltlii (Urodele Amphibian). II. Peptidases: Demonstration of sex linkage

The existence of four peptidases was demonstrated by starch gel electrophoresis in Pleurodeles waltlii: PEP-1, PEP-2, PEP-3, and PEP-4. Peptidases-3 and -4 are monomorphic, and peptidases-1 and -2 are polymorphic. The heredity of the polymorphisms was studied using individuals arising from crosses or of gynogenetic origin. Peptidase-1 is dimeric; its polymorphism depends on a pair of codominant alleles, Pep-1A and Pep-1B, which are situated on the Z and W sex chromosomes, respectively, in close proximity to, or even within, the sex differential segment. As the differential segment is very close to the centromere, the PEP-1 locus therefore also appears to be closely linked to it. Expression of the PEP-1 locus was shown to be independent of the sex hormone environment. This locus is the first case reported in amphibians of an enzyme marker linked to the genetic sex. It allows the sex of Pleurodeles to be determined before they reach sexual maturity. Peptidase-2 is monomeric. Its polymorphism depends on a pair of codominant alleles on an autosomal PEP-2 locus. The high proportion of heterozygous animals in the gynogenetic offspring of females heterozygous for the PEP-2 locus indicates segregation which is independent of the centromere. Analysis of the offspring of doubly heterozygous females (i.e., for two of the loci—LDH-B, G6PDH, PEP-1, and PEP-2) shows that the four loci are independent.

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.

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.

A genetic study of various enzyme polymorphisms in Pleurodeles waltlii (Urodele, Amphibian). III. The relationship between sex-linked peptidase-1 expression and gene-dose effects

Erythrocyte peptidase-1 was analyzed by electrophoresis in various types of triploid Pleurodeles waltlii. Densitometric analysis of the zymograms showed (1) the existence of a gene-dose effect and (2) the presence of two Pep-1B alleles for one Pep-1A allele in heterozygous triploid females of biparental origin. Owing to the sex linkage of the PEP-1 locus (alleles Pep-1A and Pep-1B situated on the Z and W sex chromosomes, respectively), the results show that the sex genotype of these females is ZWW. In a particular line called series 103, the existence of a null allele was demonstrated. Densitometric analysis of females which were Pep-1A/Pep-10 (ZW), Pep-10/Pep-10 (WW), and Pep-1A/Pep-10/Pep-10 (ZWW) confirmed the gene-dose effect.

A genetic study of various enzyme polymorphisms in Pleurodeles waltlii (Urodele Amphibian). I. Lactate dehydrogenase-B and glucose-6-phosphate dehydrogenase

On starch gel electrophoresis of erythrocyte hemolysates of Pleurodeles waltlii (Urodele Amphibian), both lactate dehydrogenase-B (LDH-B) and glucose-6-phosphate dehydrogenase (G6PDH) show polymorphism that depends on a pair of autosomal codominant alleles, confirmed by analysis of gynogenesis progeny. Diploid gynogenesis results from fusion of the female pronucleus with the second polar body. The heterozygous state of a female for a given character is maintained in certain progeny when crossing-over occurs between the locus in question and the centromere. So the high proportion of heterozygotes (45.7% for LDH-B and 76% for G6PDH) indicates the high frequency of crossing-over and hence the large distance between each of the loci and the centromere.

Maternal effect and embryonic gene expression for lactate dehydrogenase B (LDH-B), glucose-6-phosphate dehydrogenase (G6PDH), and peptidase (PEP-1) during the development of Pleurodeles waltl (urodele amphibian)

The polymorphism of three enzymes [lactate dehydrogenase B (LDH-B), glucose-6-phosphate dehydrogenase, (G6PDH), and peptidase-1 (PEP-1)] in Pleurodeles waltl has allowed the expression of the corresponding loci to be followed during the development of spawnings arising from various crosses. A maternal effect lasting up to the late tail-bud stage (approx. stage 28) was shown for PEP-1. A similar situation was observed for LDH-B and G6PDH. The embryonic alleles present retarded expression: from about stage 28 for PEP-1 and G6PDH and from about stages 22 to 24 (the young tail-bud stage) for LDH-B.

In vitro induced granulosa cell differentiation: cytoskeletal modifications

IDENTIFICATION AND PROPERTIES OF RAT BRAIN KINESIN HEAVY CHAIN ASSOCIATED WITH MITOCHONDRIA. Abdeljelil JELLAM (1), Idna SURGUCHEVA (2), Vera JANCSIK (3), Dominique FILLIOL (1) and Alvaro RENDON (1). (1) U338 Biologic de la Communication Cellulaire INSERM. 5 rue Blaise Pascal Strasbourg (France), (2) A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow 119899 (USSR), and (3) Institut of Enzymology, BRC, Hungarian Academy of Science, Budapest (Hungary). Kinesin, a mechanochemical enzyme that translocate membranous organelles towards the plus end of microtubules (MT) in cells was initlially identified and purified by its nucleotide dependent binding to MT. However, immunocytochemical and morphological approaches have shown that kinesin could be associated to intracellular membranous organelles. We have used an antibody raised against the head porlion of Drosophila kinesin heavy chain to reveal the presence of this protein in membranous organelles isolated from rat brain. By using differential centdfugation and immunoblotting, we observed a 116 kDa protein that crossreact with the antibody in microsomes, synaptic vesicles and mitochondria. This protein could be solubilized from mitochondria by treatment with low salt concenlralions. The 116kr)a solubilized protein has been identified as conventional kinesin based on its peptide mapping by limited proteolysis, amino acid composition, ATPsensitive binding to MT and the stimulation by MT of its Mg2+ .ATPase activity. These observations show that kinesin is peripherally associated to rat brain mitochondrial outer membrane; they are consistent with the hypothesis that in situ neuronal kinesin is primarily associated with membranous organelles.