Quantification of dystrophin immunofluorescence in dystrophinopathy muscle specimens.

AIMS: Duchenne muscular dystrophy (DMD) is usually associated with absent or nearly absent dystrophin expression at the sarcolemmal membrane. Quantification of very low levels of dystrophin signal in immunofluorescent studies of muscle biopsy sections presents a technical challenge. This is particularly true in the setting of proof-of-principle drug trials, in which the detection and quantification of what may be significant changes in levels of expression is important, even if absolute dystrophin levels remain low. METHODS: We have developed a method of image analysis that allows reliable and semi-automated immunofluorescent quantification of low-level dystrophin expression in sections co-stained for spectrin. Using a custom Metamorph script to create a contiguous region spectrin mask, we quantify dystrophin signal intensity only at pixels within the spectrin mask that presumably represent the sarcolemmal membrane. Using this method, we analysed muscle biopsy tissue from a series of patients with DMD, Becker muscular dystrophy, intermediate muscular dystrophy and normal control tissue. RESULTS: Analysis of serial sections on multiple days confirms reproducibility, and normalized dystrophin:spectrin intensity ratios (expressed as a percentage of normal control tissue) correlate well with the dystrophin expression levels as determined by Western blot analysis. CONCLUSION: This method offers a robust and reliable method of biomarker detection for trials of DMD therapies.

Drosophila UNC-13 is essential for synaptic transmission.

The UNC-13 protein family has been suggested to be critical for synaptic vesicle dynamics based on its interactions with Syntaxin, Munc-18 and Doc 2alpha. We cloned the Drosophila homolog (Dunc-13) and characterized its function using a combination of electrophysiology and ultrastructural analyses. Dunc-13 contained a C1 lipid-binding motif and two C2 calcium-binding domains, and its expression was restricted to neurons. Elimination of dunc-13 expression abolished synaptic transmission, an effect comparable only to removal of the core complex proteins Syntaxin and Synaptobrevin. Transmitter release remained impaired under elevated calcium influx or application of hyperosmotic saline. Ultrastructurally, mutant terminals accumulated docked vesicles at presynaptic release sites. We conclude that Dunc-13 is essential for a stage of neurotransmission following vesicle docking and before fusion.

Genetic and molecular characterization of P element-induced mutations reveals that the Drosophila ovarian tumor gene has maternal activity and a variable null phenotype.

The mutations in the ovarian tumor (otu) gene arrest oogenesis at several stages in development. A series of deletion mutations in the otu region were characterized, each of which causes the absence or reduction of the otu transcript. These alleles range from the most severe class, which results in ovaries lacking egg cysts, to relatively mild mutations that allow the development of late stage oocytes. Heteroallelic combinations of these mutations demonstrate that the phenotypic complexity of otu mutant ovaries is due to a dosage dependent requirement for otu activity. Reciprocal cross and developmental Northern blot studies suggest a maternal requirement for otu in the development of the female germline. In addition we demonstrate that the otu zygotic null phenotype is variable, ranging from the absence of cysts in the most extreme cases, to the presence of tumorous egg chambers.

Developmental analysis of the ovarian tumor gene during Drosophila oogenesis.

Severe alleles of the ovarian tumor (otu) and ovo genes result in female sterility in Drosophila melanogaster, producing adult ovaries that completely lack egg chambers. We examined the developmental stage in which the agametic phenotype first becomes apparent. Germ cell development in embryos was studied using a strategy that allowed simultaneous labeling of pole cells with the determination of embryonic genotype. We found that ovo- or otu- XX embryonic germ cells were indistinguishable in number and morphology from those present in wild-type siblings. The effects of the mutations were not consistently manifested in the female germline until pupariation, and there was no evidence that either gene was required for germ cell viability at earlier stages of development. The requirement for otu function in the pupal and adult ovary is supported by temperature-shift experiments using a heat-inducible otu gene construct. We demonstrate that otu activity limited to prepupal stages was not sufficient to support oogenesis, while induction during the pupal and adult periods caused suppression of the otu mutant phenotype.

A P element containing suppressor of hairy-wing binding regions has novel properties for mutagenesis in Drosophila melanogaster.

P elements are widely used as insertional mutagens to tag genes, facilitating molecular cloning and analyses. We modified a P element so that it carried two copies of the suppressor of Hairy-wing [su(Hw)] binding regions isolated from the gypsy transposable element. This transposon was mobilized, and the genetic consequences of its insertion were analyzed. Gene expression can be altered by the su(Hw) protein as a result of blocking the interaction between enhancer/silencer elements and their promoter. These effects can occur over long distances and are general. Therefore, a composite transposon (SUPor-P for suppressor-P element) combines the mutagenic efficacy of the gypsy element with the controllable transposition of P elements. We show that, compared to standard P elements, this composite transposon causes an expanded repertoire of mutations and produces alleles that are suppressed by su(Hw) mutations. The large number of heterochromatic insertions obtained is unusual compared to other insertional mutagenesis procedures, indicating that the SUPor-P transposon may be useful for studying the structural and functional properties of heterochromatin.

Genetic studies in Drosophila: vesicle pools and cytoskeleton-based regulation of synaptic transmission.

Presynaptic plasticity mechanisms rely on modulation of the synaptic vesicle fusion machinery and the regulated mobilization of synaptic vesicles at the active zone. This review discusses recent evidence suggesting that the relative proportions of synaptic vesicles in the reserve and ready releasable pools is the primary determinant of synaptic transmission strength, and that transport of vesicles between these pools is mediated by cytoskeletal mechanisms. Recent efforts to identify the molecules required for regulation of the presynaptic cytoskeleton suggest that common mechanisms may exist to regulate synaptic vesicle pools in widely divergent neuronal types, ranging from synaptic modulation at the Drosophila neuromuscular junction to the synaptic plasticity required for learning and memory in the mammalian brain.

Abnormal sperm-mucus penetration test predicts low in vitro fertilization ability of apparently normal semen.

OBJECTIVE: To investigate whether Kremer's sperm-mucus penetration test may predict sperm fertilizing ability in IVF. DESIGN: Kremer's test was prospectively performed on semen samples used for 66 consecutive IVF trials and compared with the fertilization rates and fertilization failure rates observed. RESULTS: Fertilization rates were significantly reduced in cases of abnormal Kremer's test (42% versus 51%; n = 745 oocytes with a statistically insignificant increase in fertilization failure rates (21% versus 10%; n = 66 trials). For abnormal semen, fertilization rates (39% versus 39%; n = 208 oocytes) and fertilization failure rates (20% versus 28%; n = 17 trials) were similar regardless of Kremer's test result. For normal semen, an abnormal Kremer's test implied a significant decrease in fertilization rates (44% versus 54%; n = 537 oocytes) with a statistically insignificant increase in fertilization failure rates (21% versus 6%; n = 49 trials). CONCLUSIONS: Abnormal Kremer's test results identify patients with a decreased in vitro fertilizing ability despite apparently normal semen samples and a group with very low fertilizing failure risk in case of normal semen samples and normal Kremer's test. Kremer's test does not add any predictive value to sperm analysis in the case of abnormal semen samples. These observations point out the importance of the male factor in fertilization failure even in the case of normal semen analysis.

[Update on ovarian hyperstimulation syndrome]. / Le point sur le syndrome d'hyperstimulation ovarienne.

The ovarian hyperstimulation syndrome (OHSS) is the most important complication of the pharmacological ovulation induction. Exclusively postovulatory, it is clinically characterized by a massive ovarian enlargement associated with an acute third-space fluid shift responsible for the development of ascites, and sometimes pleural and/or pericardial effusion. While mild OHSS has no consequence, severe forms can be life-threatening because of associated hemodynamic troubles. The main risk factors are the polycystic ovarian syndrome and an explosive ovarian response to the stimulation characterized by high serum oestradiol levels and an increased number of follicles. Ultrasound and endocrine monitoring make prevention measures possible, mainly by either abandoning the stimulation cycle or, during in vitro fertilization, cryopreserving the embryos for subsequent replacement in another cycle. Treatment consists in correcting circulatory and electrolyte imbalance. Paracentesis is more and more systematically proposed in the severe forms.

[Current program of in-vitro fertilization at the Erasmus Hospital: initial results and original ethical aspects]. / Nouveau programme de fécondation in vitro à l'hôpital Erasme: premiers résultats et aspects éthiques originaux.

The clinical results including all in vitro fertilization (IVF) cycles with oocyte pick-up in 1990 are presented. Different types of treatment including classical IVF and embryo transfer, laparoscopic replacement of zygotes in the fallopian tube (ZIFT), IVF with donor sperm (IVF-D), cross fertilization test, embryo freezing, oocyte donation and IVF with epididymal sperm were performed. The total pregnancy rate obtained reaches 38% per oocyte pick-up, 30% of clinical pregnancies (including 4 pregnancies obtained with frozen and thawed embryos). The anticipated "Take Home Baby Rate" will be around 25% per oocyte pick-up, 26 of these 40 pregnancies being today over 20 weeks of gestation. Particular ethical aspects of the program are presented: a study on couple's attitudes regarding embryo freezing as well as the final destination of possibly remaining supernumerary embryos will stress the importance of a precise clear decision on that matter before entering IVF treatment. Indeed the couple's idea on embryo destiny were very precise but also very different. The oocyte donation program has the originality of preserving the donor's anonymity by exchanging the donors recruited by the patients. It will be stressed that this kind of approach combines higher pregnancy chances for the patients, respect of ethical principles linked to gamete donation and gives satisfaction to the patients. The global normalized pregnancy cumulative curve shows that 60% of the couples entering IVF treatment will obtain a child within the first three pick-up cycles.

[Oocyte donation: ethical aspects related to the donor]. / Don d'ovocyte: aspects éthiques liés à la donneuse.

This paper analyses the reasons that brings couples to apprehend very differently the oocyte and sperm donation and highlights the difficulty of donor recruitment in those programs. The various types of donors (occasional, relational, IVF patient and professional) are described with their motivations, resistances, advantages and disadvantages. The contradictory consequences between free or paid donation, the particular risks of eggs donation (in comparison to sperm donation) for the donor as for the recipient are also highlighted. The problem of anonymity is also analysed in ethical terms but also in terms of technical efficacy: it is shown that anonymity, by authorizing sharing oocytes between recipients increases treatment efficacy by avoiding wasting this rare and precious resource of oocytes offered as a donation.

The Drosophila ovarian tumor gene is required for the organization of actin filaments during multiple stages in oogenesis.

The ovarian tumor gene is required during both early and late stages of oogenesis. Mutations produce a range of phenotypes, including agametic ovarioles, tumorous egg chambers, and late stage oogenic arrest. We demonstrate that each of these phenotypes is associated with specific aberrations in actin distribution. In the earliest case, ovarian tumor mutations cause actin filaments to accumulate ectopically in the fusome. This correlates with abnormal fusome morphology and arrested germ cell development in the germaria. Similarly, ovarian tumor function is required for the localization of actin that is essential for the maturation of ring canals. This defect gives rise to tumorous egg chambers in which germ cell numbers and morphology are profoundly aberrant. We also confirm that ovarian tumor is required for the formation of the nurse cell cytoplasmic actin array that is essential for the nonspecific transport of cytoplasmic contents to the oocyte during late oogenesis. Our data suggest that at this stage ovarian tumor controls the site where actin filaments initiate. Taken together, these studies suggest that the diverse ovarian tumor mutant phenotypes derive from the mislocalization of actin filaments, indicating a role for this gene in organizing the female germline cytoskeleton, and that the misregulation of actin can have profound effects on germ cell division and differentiation.

Comparative auto-controlled study between swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization.

This study compared swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization. Sixty trials of in-vitro fertilization (IVF), 38 with normal semen and 22 with abnormal semen, comprising 734 oocytes were included in the study. Each semen sample was prepared by both a swim-up technique and a simplified discontinuous (50%, 70%, 90%) Percoll gradient. The oocytes for each trial were distributed at random between the two sperm preparations and incubated with the same number of motile spermatozoa. Percoll gradient preparation produced a significantly higher final concentration of spermatozoa than swim-up preparation (mean +/- SEM: 6.6 +/- 1.5 x 10(6)/ml versus 1.9 +/- 0.2 x 10(6)/ml; P less than 0.01) but a significantly lower sperm motility (69 +/- 2% versus 94 +/- 1%; P less than 0.001) and a lower number of normal forms (55 +/- 2% versus 64 +/- 2%; P less than 0.01). The ability of the Percoll gradient method to extract motile spermatozoa was higher than that of the swim-up technique (20 +/- 15.6% versus 0.8 +/- 13.6%). Nevertheless, the rates of fertilization (61%), fertilization failure (18%) and polyspermia (9%), embryo quality evaluated by mean embryo scores (3.8 +/- 0.3) and the mean number of spare embryos frozen per trial (1.4 +/- 0.3) were strictly identical in both groups. The 24 pregnancies (including three from frozen--thawed embryos) obtained in these 60 trials (40% per oocyte retrieval) could not be separated according to the sperm preparation method, as embryos from both groups were replaced together.(ABSTRACT TRUNCATED AT 250 WORDS)

Oocyte shortage for donation may be overcome in a programme with anonymous permutation of related donors.

Difficulty in recruiting donors plays a crucial role in oocyte donation programmes. We report here an original strategy to overcome this problem, consisting of sharing out anonymous oocytes between recipients, which has markedly improved treatment efficiency by avoiding freezing or destruction of supernumerary embryos. Each patient received part of the supply of oocytes from several donors in four successive cycles, while each donor underwent oocyte retrieval only once. This strategy led to one pregnancy per treated donor, increased treatment efficacy by 300%, and reduced dramatically the oocyte shortage at our centre. The system avoids wastage of the rare and precious donated oocytes, increases the chance of pregnancy for each related recipient and even permits the inclusion of some recipients without related donors.

The ovarian hyperstimulation syndrome in in-vitro fertilization: a Belgian multicentric study. II. Multiple discriminant analysis for risk prediction.

The considerable overlap of distributions of values for different parameters between control and ovarian hyperstimulation syndrome (OHSS) populations makes any single variable inefficient for risk prediction. Combinations of variables were studied in a discriminant function in order to increase predictivity and decrease the false negative rate. Such analyses were performed on two groups of in-vitro fertilization (IVF) patients: all OHSS cases (n = 128) (group A) and only severe OHSS cases (n = 92) (group B). Progressive introduction and automated stepwise selection of variables were applied to both groups. The best prediction (78.5%) was obtained in group A under post-oocyte retrieval conditions using log oestradiol, slope of log oestradiol increment, human menopausal gonadotrophin (HMG) dosage, number of oocytes retrieved and ratio of luteinizing hormone/follicle stimulating hormone (LH/FSH), in the formula. The corresponding false negative rate was 18.1%. However, effective prevention of OHSS implies the ability to withhold human chorionic gonadotrophin injection. Therefore a formula for pre-oocyte retrieval conditions was established yielding a prediction rate of 76.1% with a false negative rate of 18.1%. To be validated, such formulae have to be applied to another population of IVF cases used as a 'testing-set'.