Genetic counseling for cystic fibrosis: A basic model with new challenges.

While the goals of genetic counseling for cystic fibrosis - delivering relevant information on the risk of recurrence and nondirectional support of couples at risk in their reproductive choices - have not changed fundamentally, the practice has evolved considerably in the last decade, growing more complex to face new challenges but also proving more effective. Many factors have contributed to this evolution: technical progress in the exploration of the genome (new generation sequencing) and in reproductive medicine, but also societal developments promoting access to genetic information and the professionalization of genetic counselors in France. The prospect of expanded pre-conception screening of at-risk couples makes genetic counselors major actors not only in medical care centers, but also in modern society by contributing to genetic education among citizens. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.

Effects of phorbol esters on steroidogenesis in small bovine luteal cells.

The possible influence of an activator of protein kinase C, the tumor-promoting phorbol ester, PMA (phorbol-12-myristate-13-acetate), upon small bovine luteal cell steroidogenesis was investigated in vitro, PMA had no significant effect on basal and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production but markedly modulated the LH-stimulated progesterone and cAMP productions. PMA potentiated the LH-stimulated cAMP accumulation whatever the dose of LH used. It also potentiated the LH-induced progesterone production in the presence of low doses of LH. Paradoxically, in the presence of maximal or submaximal effective doses of LH, PMA exerted a time- and dose-dependent inhibition of progesterone synthesis. Diacylglycerol was able to mimic the effects of PMA on LH-induced steroidogenesis. These observations suggest that the Ca2+- and phospholipid-dependent protein kinase C can modulate the regulation by LH of small bovine luteal cell steroidogenesis at a step before the synthesis of cAMP. They also suggest that the interaction between LH and its receptor is able to trigger a negative regulatory signal which would be only expressed for high doses of LH and in the presence of an activator of PKC.

Alternative splicing events in the coding region of the cytochrome P450 aromatase gene in male rat germ cells.

Expression of cytochrome P450 mRNA in rat germ cells was characterized by reverse transcription PCR with various primers located at the 3'-end of the coding region. At least two unusual isoforms (Ex10-S and INT) of P450 aromatase (P450arom) mRNA were expressed. Analysis of their sequences demonstrated that an alternative splicing event occurred first at the exon-intron boundary of the GT consensus sequence of the last coding exon, and second in the internal 5' donor inside exon 9 used as a minor cryptic splicing site. These isoforms lacked the last coding exon which contained the heme-binding domain; in addition, for the Ex10-S transcript, the catalytic domain was also absent because of a frameshift in the open reading frame. The deduced amino acid sequences led to truncated P450arom polypeptides without the heme-binding domain, which were probably unable to convert androgens into estrogens. Adult rat germ cells are able to express P450arom mRNA, which is then translated into a biologically active enzyme which is involved in estrogen production. Moreover, for the first time, we report the existence of alternative splicing events of P45Oarom mRNA in pachytene spermatocytes and round spermatids, which probably cannot encode functional aromatase molecules.

Presence of exon I.4 mRNA from CYP19 gene in human granulosa cells.

Estrogens are synthesized from C19 steroids by a unique form of cytochrome P450 aromatase. Expression of the human CYP19 gene involves tissue specific use of alternative promoters. In the present study, an RT-PCR procedure was used to amplify and quantify various transcripts expressed in human granulosa cells. Cells were aspirated together with follicular fluid from Periovulatory ovarian follicles present in ovaries of 14 patients undergoing a treatment for in vitro fertilization. Sequencing of PCR products demonstrated the presence of exon I.4-specific transcripts in addition to exon P.II, exon I.3 and I.3-truncate transcripts. Quantitative results confirmed that exon P.II specific transcripts were largely predominant compared to other exon-specific transcripts, and that exon I.4-specific transcripts were the least abundant.

Involvement of the phospholipase C second messenger system in the regulation of steroidogenesis in small bovine luteal cells.

We have previously suggested that the interaction between luteinizing hormone (LH) and its receptor, in addition to stimulating adenylate cyclase, is able to trigger a negative regulatory signal at a step beyond cAMP synthesis (Benhaim et al. (1987) FEBS Lett. 223, 321-326). The present study was conducted to determine whether the phospholipase C system is involved in this phenomenon. Small bovine luteal cells from pregnant cows were incubated with phospholipase C, A23187, an ionophore of calcium and/or phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), in the presence or absence of bovine luteinizing hormone or dibutyryl cyclic AMP (dbcAMP). A23187 associated with PMA was able to mimic the stimulatory effect of phospholipase C on basal progesterone production, whereas neither A23187 nor PMA alone had any effect. In the presence of high doses of LH, phospholipase C inhibited progesterone and cAMP production in a dose-dependent manner. A23187 and PMA were able to mimic the inhibition of progesterone synthesis but stimulated LH-induced cAMP accumulation. When cells were stimulated by high doses of dbcAMP, phospholipase C and A23187 but not PMA inhibited progesterone synthesis. These observations suggest that (1) phospholipase C can mimic the post-cAMP negative regulatory signal induced in vitro by high doses of LH, in the presence of an activation of PKC; (2) phospholipase C is also able to mimic in vitro the luteolytic properties of prostaglandin F2 alpha that we previously described (Benhaim et al. (1987) Prostaglandins 33, 227-239); and (3) under basal conditions or in the presence of low doses of LH, the phospholipase C system slightly stimulates steroidogenesis.

Quantification of cytochrome P450 aromatase transcripts before and during luteal phase in rabbit.

The aim of the present study was to quantify the promoter II- and I.r-derived transcripts of p450 aromatase gene during follicular stages and during corpus luteum formation in the rabbit. An ovulatory dose of hCG induced, first the disappearance of 90% of aromatase transcripts since 6 h before ovulation, and second a gradual decrease during pseudopregnancy. Individual quantification of both the promoter-derived transcripts showed that promoter II-derived transcript was the main transcript expressed both during follicular phase and pseudopregnancy, but kinetics of disappearance were not similar between both the promoter-derived transcripts. Moreover, hCG up-regulates aromatase expression in vitro in luteal tissue but estradiol, which was without effect on aromatase expression in preovulatory granulosa cells, down-regulates this expression in luteal tissue. In conclusion, the regulation of P450 aromatase in rabbit is mainly under control of promoter II regardless of which cyclic stage is studied. Moreover, we reported an opposite effect of estradiol on aromatase expression in vitro between follicular and luteal cells.

The possible involvement of LH/hCG induced mitochondrial proteins in the regulation of steroidogenesis in bovine luteal cells.

In previous studies we described the synthesis of three mitochondrial proteins (A, B and C) in response to acute in vitro stimulation by lutropin of small bovine luteal cells. Protein A had a molecular weight of 28 kDa and an isoelectric point (pI) of 6.7. Proteins B and C had a molecular mass of 27 kDa and pI of 6.2 and 6.4, respectively. The appearance of these proteins was prevented by 100 microM cycloheximide. In the present study, we have shown that the time course of synthesis of protein A and its hCG dose-response closely parallel the increase in progesterone production. The induction by hCG of protein A was already observed after a 5 min incubation. Pulse chase experiments by addition of excess unlabelled methionine after prelabelling with [35S]methionine indicated that its half-life was approximately 15-20 min. Study of 32P labelled phosphate incorporation into individual proteins and treatment by alkaline phosphatase of [35S]methionine-labelled proteins demonstrated that none of the three proteins A, B or C was a phosphoprotein. Localization of protein A in mitochondria, at the site of the rate limiting step in steroidogenesis, and the high degree of correlation between its 35S labelling and progesterone production argue in favour of its involvement in the acute regulation of steroidogenesis.

Expression of two aromatase cDNAs in various rabbit tissues.

Aromatase is a steroidogenic enzyme complex which catalyses the conversion of androgens to estrogens. In a previous study, we elucidated the structure of a 2.9 kb aromatase cDNA from ovarian rabbit tissue. We report here, the structure of another shorter aromatase cDNA (1.5 kb) from the same tissue. This cDNA is likely to encode for a nonfunctional aromatase which would lack an heme binding domain. We have shown using an RT-PCR technique that rabbit placental tissue, like the ovarian one, expresses both the 2.9 and 1.5 kb cDNA and that the adipose tissue expresses the 2.9 kb cDNA. Using a 5' RACE procedure, we obtained the 5' end of the placental transcripts. Comparison of its sequence with the 5' end of the ovarian one suggests the existence of distinct exon 1 sequences for each one of the two tissues as already described in the human. These results point to the rabbit as a useful laboratory animal for studying regulation of aromatase expression in adipose and placental tissues.

Involvement of theca cells and steroids in the regulation of granulosa cell apoptosis in rabbit preovulatory follicles.

Follicular atresia is characterized by a rapid loss of granulosa cells and, to a lesser extent, theca cells, via apoptosis. The aim of this study was to investigate the possible involvement of theca cell secretions in the regulation of apoptosis of rabbit granulosa cells. The annexin-V binding method based on externalization of phosphatidylserine to the outer layer of plasma membrane during apoptosis was used to detect apoptotic granulosa cells in flow cytometry. Regulation of apoptosis of granulosa cells was studied in three different culture systems: (i) isolated cultured granulosa cells, (ii) granulosa cells obtained from cultured preovulatory follicles and (iii) granulosa cells co-cultured with theca cells. The results of this study indicate that: (i) the rate of apoptosis of granulosa cells was significantly reduced when granulosa cells were co-cultured with theca cells or obtained from cultured preovulatory follicles in comparison with isolated cultured granulosa cells; (ii) FSH exerts its anti-apoptotic effect only on granulosa cells issued from cultured preovulatory follicles; (iii) ovarian steroids do not affect the percentage of isolated apoptotic granulosa cells; and (iv) the occurrence of an apoptotic process in rabbit theca cells could be upregulated in vitro by hCG and an analogue of the gonadotrophin second messenger cAMP. The results of this study indicate that in rabbits (i) steroids were ineffective in vitro in protecting isolated granulosa cells against apoptosis in comparison with observations in vivo in rats, and (ii) the presence of theca cells was efficient to reduce granulosa cell apoptosis but not sufficient to allow the anti-apoptotic effect of gonadotrophins observed in cultured follicles.

Acute stimulation by lutropin of mitochondrial protein synthesis in small luteal cells.

A two-dimensional electrophoretic technique was used to study the effect of acute stimulation of bovine luteal cells with lutropin on protein synthesis. Cells were incubated for 30 min with [35S]methionine in the presence of stimulating levels of luteinizing hormone (lutropin), after which the proteins were analyzed by autoradiography. Lutropin or N6,2'-O-dibutyryl-adenosine 3',5'-phosphate (Bt2cAMP) induced the labelling of three proteins, referred to as proteins A, B and C. Protein A, had a molecular mass of 28 kDa and an isoelectric point (pI) of 6.7. Proteins B and C had a molecular mass of 27 kDa and pI of 6.2 and 6.4 respectively. After subcellular fractionation, the three proteins were found to be markedly concentrated in the only fraction enriched in an established mitochondrial marker. Moreover, protein A was one of the major mitochondrial newly synthesized proteins. Its appearance was observed after a 5-min incubation and was prevented by 100 microM cycloheximide. The acute accumulation of proteins A, B and C in mitochondria, the site of the rate-limiting step of steroidogenesis, suggest that they could be involved in the mechanism of stimulation by lutropin of progesterone synthesis.

In vitro action of PG F2 alpha on progesterone and cAMP synthesis in small bovine luteal cells.

The action of prostaglandin F2 alpha (PG F2 alpha) on incubated small bovine luteal cells in the presence or in the absence of bovine luteinizing hormone (LH) or dibutyryl cyclic adenosine monophosphate (db cAMP) was investigated. In the absence of LH and db cAMP, PG F2 alpha stimulated progesterone synthesis at concentrations of 10 ng/ml and 100 ng/ml but had no effects at concentrations below 1 ng/ml. PG F2 alpha partially inhibited the LH or db cAMP stimulated progesterone synthesis. This inhibition was maximal for PG F2 alpha concentrations around 100 pg/ml whereas distinctly higher or lower concentrations were without effect. At the concentration of 100 pg/ml, PG F2 alpha partially inhibited the LH induced cAMP accumulation. These results demonstrate an "in vitro" action of PG F2 alpha on bovine luteal cells. They indicate that the luteolytic action of PG F2 alpha in the bovine species could involve, as already suggested for the rat, both an inhibition of the LH induced synthesis of cAMP and an inhibition of the action of cAMP.

Rapid sequencing of rabbit aromatase cDNA using RACE PCR.

The sequencing of aromatase cDNA from rabbit granulosa cells was obtained by RACE PCR. This cDNA is 2.9 kb long. The first 119 nucleotides correspond to the first untranslated exon. Nucleotides 120 to 1,629 correspond to the coding region (1,509 nucleotides) and the rest of the sequence is non coding and contains a polyadenylation signal. Translation of the cDNA sequence indicates that the protein is composed of 503 amino acids, like in human aromatase. Its molecular weight is 57.4 kDa. The alignment between the rabbit aromatase amino acid sequence and other aromatases already described in the human, mouse, rat, cow, pig, chicken, rainbow trout and teleost fish shows that the rabbit protein exhibits the highest homology with the human one (85%).

Expression and immunolocalization of functional cytochrome P450 aromatase in mature rat testicular cells.

Aromatase activity has been measured in Leydig cells and Sertoli cells from both immature and mature rats. Cytochrome P450 aromatase (P450arom) has been immunolocalized in germ cells of the rodent, bear, and rooster. Our purpose was to investigate expression of and to immunolocalize P450arom in adult rat testicular cells. After Western blotting with a specific anti-cytochrome P450arom antibody, we demonstrated the presence of a 55-kDa protein in mature rat seminiferous tubules and crude germ cell preparations. Immunoreactive aromatase was detected both in cultured rat Leydig cells and in testis sections (interstitial tissue and elongated spermatids showed positive immunoreactivity for P450arom). We next used reverse transcription-polymerase chain reaction to localize and quantify the P450arom mRNA in the various testicular cells. In rat Leydig cells, the amount of P450arom mRNA was 15 times higher than in Sertoli cells (34.1+/-3.2 to 2.3 +/-0.2 x 10(-3) amol/10(6) cells, respectively). In pachytene spermatocytes, round spermatids, and testicular spermatozoa the P450arom mRNA levels were 38.7+/-8.1, 20.4+/-3.8, and < 1.3 x 10(-3) amol/10(6) cells, respectively. The aromatase activity was 2.5-4 times higher in testicular spermatozoa (8.48+/-1.98 fmol/10(6) cells per hour) than in other germ cells. These results indicate that in mature rats, not only Leydig cells and Sertoli cells but also germ cells have the capacity to express functional P450arom. According to the germ cell maturation state, there was an inverse relationship between P450arom mRNA content and the biological activity of the protein. The expression of the functional P450arom in mature rat germ cells confirms the existence of an additional source of estrogens within the genital tract of the male.

Expression of the rabbit cytochrome P450 aromatase encoding gene uses alternative tissue-specific promoters.

The aim of the present study was to analyse the tissue-specific expression of various promoter-derived transcripts from the gene encoding rabbit aromatase cytochrome P450. A new promoter, named I.r, was identified, and promoters II and I.r were sequenced. Promoter I.r-derived transcripts were found in preovulatory granulosa cells, corpus luteum, placenta and adipose tissue. An alternative splice variant of this transcript was found with tissue-specific preference. Tissue-specific expression of promoter-derived variants was studied in the ovary before and after ovulation. While the level of promoter II-derived transcript decreased dramatically after ovulation, that of promoter I.r-derived transcript remained unchanged, indicating that promoter II and promoter I.r were not controlled by a single regulation system. The existence of this dual system of regulation suggests that the rabbit ovary could be a useful model to study the promoter-specific regulation of aromatase.

Estrogen response of MCF-7 cells grown on diverse substrates and in suspension culture: promotion of morphological heterogeneity, modulation of progestin receptor induction; cell-substrate interactions on collagen gels.

In this study we observed the incidence of hormone sensitivity in the response of MCF-7 cells to estrogen stimulation when the cells were cultured in different contact environments (hydrophilic plastic, bovine corneal extracellular matrix, type I collagen and in suspension culture). The major purpose was to describe the influence of cell to cell and cell to substrate contacts on the morphological response to estrogen treatment. However, other parameters including growth and induction of progestin receptor were also explored, keeping in mind that the MCF-7 cell line, although representative of normal mammary epithelium in that it contains a similar hormone receptivity, was selected in vitro from a metastatic population in a pleural effusion. Although substrate conditions did not modify growth enhancement by estrogens, progestin receptor levels were significantly higher in three-dimensional spheroid cultures in which cell to cell contacts were optimal due to elimination of basal contact. A careful morphological survey of large surfaces lead to an objective opinion of the overall effect of the hormone treatment on the non-cloned cell line in which a marked heterogeneity in the response of individual cells was observed. In terms of morphofunctional differentiation, the edification of acini with dense microvillus coating was best in suspension culture. When sections were made perpendicular to the plane of cultures on collagen gel rafts two other phenomena were noted: decrease in intercellular junctions, resulting in reduced cell to cell cohesion, and accumulation biodegradation products in the collagen lattice. This suggested a hormone-mediated interaction between the metastatic cells and the fibrillar substrate, collagen I, one of the major constituents of tissue stroma. This estrogen response might be related to the metastatic phenotype and must be distinct from their hormone sensitivity in terms of growth and differentiation since hormone receptivity is generally considered to be a favorable prognosis for breast cancer.

Estradiol membrane binding sites on human breast cancer cell lines. Use of a fluorescent estradiol conjugate to demonstrate plasma membrane binding systems.

A fluorescent estradiol macromolecular complex was used to study and to characterize steroid binding to membranes of living target cells. Ligand binding to plasma membranes was quantitated with a sensitivity of 0.1 nM. In this way, we found two types of estradiol-binding sites on hormone sensitive MCF-7 cells. Type A sites (8000-16000 sites per cell) were rapidly saturated at low concentrations of the estradiol-bovine serum albumin-fluorescein isothiocyanate macromolecular complex (E2-BSA-FITC). They had a greater affinity for the complex than did the type B sites for which a phenomenon of cooperative fixation was shown. The complex binding was displaced by estrogenic molecules, but not by non-estrogenic compounds, such as cortisol or progesterone. We also studied complex binding on another breast cancer cell line, MDA-MB-231 (MDA), without intracellular estrogen receptors. These cells showed a specific plasma membrane binding system for estrogen, but lacked the high affinity type A binding site. Then, we report the effects of enzyme treatments (trypsin, phospholipase A2 and neuraminidase) on E2-BSA-FITC binding to MCF-7 cell membranes. The quantity of complex bound to membranes decreased after phospholipase and neuraminidase treatments and increased after trypsin. But, in the three cases, the binding was no longer specific because it could not be displaced by E2-BSA or by estradiol. The enzymatic effects were reversible and specific binding was totally restored within 24 h. However, in the presence of the protein synthesis inhibitor, cycloheximide, no restoration of specific binding occurred on trypsin-treated cells. Estrogen binding to MCF-7 and MDA cell plasma membranes thus possesses the three characteristics of all mediated transport processes across biological membranes: saturability, substrate specificity, and specific inhibition. However, the high affinity type A binding site was found only on the estrogen-sensitive cell line, MCF-7.

Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.

We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.