Ocular tissue interactions during the development of human fetal neuroretina grafted into nude mice

To determine the roles of different ocular tissues in the development of the human fetal neuroretina, a study ethically and technically impossible in human subjects, human embryonic and fetal retinas were heterotopically implanted into nude mice. Ninety-five eyeballs were obtained from legally aborted 6- to 7-week-old embryos or 8- to 10-week-old fetuses. Ten isolated neuroretinas with vitreous but without pigment epithelium, 20 half-eyeballs and 70 intact eyeballs, of which 12 had a thick layer of periocular tissue, were microsurgically grafted. Five intact eyeballs were used for reference. Over a period of 1-245 days, all of the grafts were removed for light and electron microscopy observations. All of the isolated neuroretinas had disappeared by the second day after transplantation. Grafts of the posterior section of the eyeball contained only some clusters of pigment epithelium, occasionally covered with undifferentiated neuroretinal cells. Grafts of the retrolental section of the eyeball contained small areas of dysplasic neuroretina with folds and rosettes. Grafts of the 70 intact eyeballs were successful, but only 26 showed normal histological organization of the choriocapillaris, the retinal pigment epithelium and the neuroretina in the posterior part of the posterior chamber. Photoreceptor differentiation was evident in these retinas after approximately 80 days of transplantation and was complete after 166 days. Their anterior part was always dysplasic, with occasional ciliary differentiation. Twenty-three grafted eyeballs had a dysplasic neuroretina with folds, rosettes and necrotized areas. Twenty-one were atrophic, 12 of which were the eyeballs grafted with periocular tissue. These results demonstrate the role of the fetal mesenchyme and pigment epithelium in the rapid revascularization, and subsequent survival and tissue organization, of the neuroretina. The stratified development of the neuroretina required a thin mesenchymal environment for revascularization of the graft by human vasculogenesis or neoangiogenesis and a normal retinal pigment epithelium for normal neuroretinal differentiation. When these conditions were not satisfied, the neuroretina disappeared or was dysplasic, partly necrotized or atrophic. This model might prove useful for a number of therapeutic or clinical studies.     

Effect of follicular size on meiotic and developmental competence of porcine oocytes

In several species, the developmental competence of the oocyte is acquired progressively during late follicular growth, after the acquisition of the competence to resume and complete meiosis. In the pig, full meiotic competence of the oocyte is reached in ovarian follicles with a diameter of 3 mm or more. However, there is no information about developmental competence acquisition. We analyzed the ability of oocytes from three foll icular size classes to resume and complete meiosis, to be fertilized, and to develop in vitro to the blastocyst stage. A total of 941 follicles were dissected from slaughterhouse gilt ovaries and classified as small (<3 mm, n = 330), medium (3-5 mm, n = 373), or large (>5 mm, n = 238). The cumulus-oocyte complexes recovered from these follicles were submitted to in vitro maturation for 44 h in TCM199 supplemented with 10 ng/ml EGF, 400 ng/ml pFSH and 570 microM cysteamine; in vitro fertilized for 18 h in mTBM with 10(5) frozen-thawed percoll-selected sperms/ml; and developed for 7 days in mSOF. Samples of oocytes or presumptive zygotes were fixed and stained at the end of maturation and fertilization. Groups of oocytes were cultured for 3 h in the presence of 35S-methionine before or after maturation for SDS-PAGE analysis of protein neosynthesis. More oocytes originating from medium and large follicles were competent for maturation than oocytes from small follicles (77 and 86% of metaphase II, respectively, versus 44%, P < 0.05). More oocytes from medium and large follicles werepenetratedby spermatozoa during in vitro fertilization, resulting in significantly more oocytes presenting two or more pronuclei at the end of fertilization (73 and 77% for medium and large follicles, respectively, versus 53% for small follicles, P < 0.05). More oocytes from medium and large follicles developed to the blastocyst stage (14 and 23%, respectively) than those from small follicles (3%, P < 0.05), even if the development rates were corrected by the maturation or fertilization rates. It is concluded that a high proportion of oocytes harvested from follicles of less than 3 mm in the pig are not fully competent for meiosis and are cytoplasmically deficient for development.     

Aminoglycoside-derived cationic lipids as efficient vectors for gene transfection in vitro and in vivo

Background

Cationic lipids are at present very actively investigated for gene transfer studies and gene therapy applications. Basically, they rely on the formation of DNA/lipid aggregates via electrostatic interactions between their cationic headgroup and the negatively charged DNA. Although their structure/activity relationships are not well understood, it is generally agreed that the nature of the positive headgroup impacts on their transfection activity. Thus, we have directed our efforts toward the development of cationic lipids with novel cationic moieties. In the present work, we have explored the transfection potential of the lipophilic derivatives of the aminoglycoside kanamycin A. Indeed, aminoglycosides, which are natural polyamines known to bind to nucleic acids, provide a favorable scaffold for the synthesis of a variety of cationic lipids because of their structural features and multifunctional nature.

Methods and results

We report here the synthesis of a cationic cholesterol derivative characterized by a kanamycin A headgroup and of its polyguanidinylated derivative. The amino‐sugar‐based cationic lipid is highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE). Its polyguanidinylated derivative was also found to mediate in vitro gene transfection. In addition, colloidally stable kanamycin‐cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways in vivo.

Conclusions

These results reveal the usefulness of cationic lipids characterized by headgroups composed of an aminoglycoside or its guanidinylated derivative for gene transfection in vitro and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Mitosis under control

The mitotic checkpoint is essential to ensure accurate chromosome segregation by allowing a mitotic delay in response to a spindle defect. This checkpoint postpones the onset of anaphase until all the chromosomes are attached and correctly aligned onto the mitotic spindle. The checkpoint functions by preventing an ubiquitin ligase called the anaphase-promoting complex (APC) from ubiquitinylating proteins whose degradation is required for anaphase onset. Loss of this checkpoint results in chromosome missegregation in higher eukaryotes and may contribute to the genomic instability observed in most of the tumour cells.     

Gene flow estimation with microsatellites in a Malagasy seed orchard of Eucalyptus grandis

Eucalyptus grandis has a mixed-mating reproductive system. Malagasy Eucalyptus seed orchards were established 15 years ago with two aims both based on panmixia: open-pollinated seed production and genetic improvement. The panmixia hypothesis has never been confirmed in the seed orchard. From a seedling seed-orchard stand comprising 349 trees and using data obtained with six selected microsatellite markers, paternity analysis was performed for 724 offspring collected on 30 adult trees. Paternity assignment, based on exclusion procedures and likelihood-ratio method, was achieved with high accuracy; the exclusion probability value was 0.997. The outcrossing rate was very high (96.7%). More than 50% of potential male trees (199 out of 349) in the seed orchard contributed to pollination for 440 offspring of 30 progenies (8.6% of the basic population). The pollination rate from outside the seed orchard was high (39.2%), but might be due to the small size of this seed orchard. This study showed that "panmixia-like pollination" can be assumed.