Effect of timing of oocyte denudation and micro-injection on survival, fertilization and embryo quality after intracytoplasmic sperm injection

In human in-vitro fertilization (IVF), the oocytes are surrounded by cumulus and corona cells at the time of insemination so that their maturity cannot easily be evaluated. The best IVF results are obtained if the oocytes are inseminated 2-6 h after retrieval. In the intracytoplasmic sperm injection (ICSI) procedure, the oocytes are denuded by enzymatic and mechanical treatment in order to be able to perform the injection. As a consequence, the nuclear maturity of the oocytes can be evaluated and only those that have extruded the first polar body are injected. However, metaphase-II oocytes that have not yet reached cytoplasmic maturity cannot be recognized. The purpose of this study was to investigate the effect of different timing of cumulus- corona cell removal and injection on the outcome of ICSI. For this we allowed the oocytes to complete in-vitro cytoplasmic maturation in two different culture conditions: (i) surrounded by their cumulus and corona cells or (ii) totally denuded. We performed three different studies on sibling oocytes obtained after a standardized buserelin/human menopausal gonadotrophin (HMG) protocol. We investigated the effect of early (1-2 h after retrieval) and late (5-6 h after retrieval) oocyte denudation and injection on the survival and fertilization of the injected oocytes and on embryo cleavage after fertilization. We found no statistically significant differences between early and late injection, indicating that after a standardized buserelin/HMG protocol the metaphase-II oocytes do not need time for further cytoplasmic maturation. Furthermore, a different timing of cumulus-corona cell removal has no effect on the outcome of ICSI, suggesting that the surrounding cells are not necessary for survival, fertilization and cleavage after ICSI.      

Relationship between serum follicle stimulating hormone in the male and standard sperm parameters, and the results of intracytoplasmic sperm injection

Serum follicle stimulating hormone (FSH) is routinely measured when evaluating the infertile male for intracytoplasmic sperm injection (ICSI). However, among the sperm parameters, only its relationship with sperm concentration is well documented. Few investigations concern the relationship between FSH and sperm motility and morphology, and the results of ICSI. A retrospective study of 316 couples who underwent ICSI was carried out to determine the relationships between serum FSH concentrations in the male and (i) standard sperm parameters_(concentration, motility and morphology) and (ii) fertilization, cleavage, pregnancy and implantation rates after ICSI. There was an inverse correlation with sperm concentration and total motility but no relationship was found with progressive motility and sperm morphology. Neither was any relationship found between serum FSH and fertilization, cleavage, pregnancy and implantation rates, and the results of ICSI. These findings suggest the need to review the routine measurement of serum FSH in the infertile male when ICSI is the planned treatment procedure.      

Rapid presumptive bacteriological diagnosis of Legionnaires disease.

A simple, relatively rapid silver impregnation stain has been found to stain Legionella pneumophila effectively in paraffin-embedded tissue sections while permitting visualization of histological detail. It may also be used to stain the organism in body fluids. The stain is not specific and thus must be confirmed by direct fluorescent-antibody technique or culture, but, in the absence of other bacilli demonstrable by Gram or other stain, visualization of typical bacillary forms in a patient with illness compatible with Legionnaires disease provides strong presumptive evidence supporting this diagnosis.     

Migration and distribution of circumpharyngeal crest cells in the chick embryo

The cardiac neural crest is located in a transitional area on the neuraxis between trunk and cephalic regions and gives rise to both the dorsolateral and ventrolateral crest cell populations. Around stage 18 of chick development, a mass of E/C8⁺ cells surrounds the postotic pharyngeal arches and forms a crescent-shaped arch, termed the circumpharyngeal ridge. Using immunohistochemistry and quail-chick chimeras, it was determined that the E/C8⁺ cell mass located in the circumpharyngeal ridge derives from the dorsolateral component of the cardiac neural crest. The ventrolateral cell population of the cardiac crest is located more medially and shows long-persistent HNK-1 immunoreactivity dorsolateral to the foregut. The crest cells that populate the gut arise from the caudal portion of the circumpharyngeal crest and are always located caudal to the caudalmost pharyngeal ectomesenchyme. Circumpharyngeal crest cells continuously populate the pharyngeal arch ectomesenchyme and enteric nervous system on the lateral side of the foregut wall, as well as the hypoglossal pathway which develops within the ventral portion of the circumpharyngeal ridge. E/C8 and HNK-1 immunoreactivity are associated with the cells migrating via the dorsolateral (circumpharyngeal) and ventrolateral pathways, respectively, with one exception: there is a population of putative crest cells along the proximal course of the vagal intestinal branch that shows both immunoreactivities around stage 20. Dil labeling of the cells in the circumpharyngeal ridge suggests that the cells are contributed from the circumpharyngeal ridge to this population. Thus, the distribution of the circumpharyngeal crest cells and their derivatives coincides with the peripheral branch distribution of the cranial nerves IX, X, and XII, whose development is selectively affected in the absence of the cardiac neural crest, the source of the circumpharyngeal crest.© Willey-Liss, Inc.     

Renal allograft rejection: induction and function of adhesion molecules on cultured epithelial cells.

The interaction of graft-infiltrating immune cells with donor parenchymal cells is an important early event in allograft rejection. This binding is stabilized by interaction of antigen-independent 'adhesion' molecules expressed on the two cell types. As the level of expression of these molecules can be altered during inflammation, a series of experiments was performed to examine the effects of the inflammatory cytokines interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) on adhesion molecules expressed by cultured human renal tubular epithelial cells. These cells constitutively expressed ICAM-1 and LFA-3. Incubation with IFN-gamma increased expression of ICAM-1 but had no significant effect on expression of LFA-3 (P greater than 0.05). Incubation with TNF-alpha increased expression of both ICAM-1 and LFA-3; IFN-gamma synergized with TNF-alpha to further augment expression of these molecules. Peripheral blood lymphocytes (PBL) showed an enhanced binding to allogeneic renal epithelial cell monolayers which had been pretreated with IFN-gamma or TNF-alpha. MoAbs specific for ICAM-1 or its ligand LFA-1 inhibited adhesion of PBL to either IFN-gamma- or TNF-alpha-pretreated renal cells. By contrast, antibodies specific for LFA-3 or its ligand CD2 only significantly blocked PBL adhesion to renal cells which had been pretreated with TNF-alpha. Combination of antibodies specific for multiple components of the adhesion systems produced greater inhibition of adhesion than was produced by any single MoAb. These results suggest that the inflammatory cytokines IFN-gamma and TNF-alpha up-regulate expression of functional ICAM-1 and LFA-3 molecules which can augment the binding of potentially graft-damaging lymphoid cells to renal tubular epithelial cells.     

Partial gonadal dysgenesis in a patient with a marker Y chromosome

We evaluated a patient with partial gonadal dysgenesis including a right dysgenetic testis and a left streak gonad with rudimentary fallopian tube and uterus. She had ambiguous external genitalia and was raised female. Although her height is normal (25th centile at age 12 years), she has some findings of Ullrich–Turner syndrome. Her karyotype was reported to be 46, X, + marker; subsequent molecular investigations showed the marker to be the short arm of the Y chromosome. Genomic DNA, isolated from leukocytes of the patient and her father, was digested with a variety of restriction endonucleases and subjected to Southern blot analysis. A positive hybridization signal was obtained with probes for the short arm of the Y chromosome (pRsY0.55, SRY, ZFY, 47Z, pY-190, and YC-2) in DNA from the patient, indicating the presence of most if not all of the short arm, while long arm probes (HinfA and pY3.4) indicated that at least 75% of the long arm of the Y chromosome was missing. The gene responsible for testicular determination (TDF) is on the distal portion of the short arm of the Y chromosome; Yq has no known influence on sex determination. Hence, the deletion of the long arm of the Y chromosome cannot explain the gonadal dysgenesis in this patient. One explanation for the gonadal dysgenesis and Ullrich–Turner phenotype in the patient could be undetected 45, X/46,X, + marY mosaicism but no such mosaicism was observed in peripheral lymphocytes. Several investigators have suggested the presence of an “anti-Turner” gene near TDF. Hence it is possible that the clinical phenotype in our patient results from a Y chromosomal defect in sequences flanking TDF, which reduces the function of both TDF and the “anti-Turner” genes.     

The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

A locus for autosomal dominant anterior polar cataract on chromosome 17p

Inherited cataract is a clinically and genetically heterogeneous disease. Here we report the identification of a new locus for an autosomal dominant anterior polar cataract on the short arm of chromosome 17. To map this new locus we performed genetic linkage analysis with microsatellite markers in a four-generation pedigree. After exclusion of seven candidate loci for cataract, we obtained significant positive LOD scores for markers D17S849 (Z = 4.01 / theta = 0.05) and D17S796 (Z = 4.17 / theta = 0.05). Multipoint analysis gave a maximum LOD score of 5.2 (theta max = 0.06) between these two markers. From haplotype analysis, the cataract locus lies in the 13 cM interval between markers D17S849 and D17S796. This study provides the first genetic mapping of an autosomal dominant anterior polar cataract.      

Sex determination in platypus and echidna: autosomal location of <Emphasis Type="Italic">SOX3</Emphasis> confirms the absence of <Emphasis Type="Italic">SRY</Emphasis> from monotremes

In eutherian ('placental') mammals, sex is determined by the presence or absence of the Y chromosome-borne gene SRY, which triggers testis determination. Marsupials also have a Y-borne SRY gene, implying that this mechanism is ancestral to therians, the SRY gene having diverged from its X-borne homologue SOX3 at least 180 million years ago. The rare exceptions have clearly lost and replaced the SRY mechanism recently. Other vertebrate classes have a variety of sex-determining mechanisms, but none shares the therian SRY-driven XX female:XY male system. In monotreme mammals (platypus and echidna), which branched from the therian lineage 210 million years ago, no orthologue of SRY has been found. In this study we show that its partner SOX3 is autosomal in platypus and echidna, mapping among human X chromosome orthologues to platypus chromosome 6, and to the homologous chromosome 16 in echidna. The autosomal localization of SOX3 in monotreme mammals, as well as non-mammal vertebrates, implies that SRY is absent in Prototheria and evolved later in the therian lineage 210-180 million years ago. Sex determination in platypus and echidna must therefore depend on another male-determining gene(s) on the Y chromosomes, or on the different dosage of a gene(s) on the X chromosomes.