Ocular and craniofacial phenotypes in a large Brazilian family with congenital aniridia.

Congenital aniridia is a rare genetic disorder characterized by varying degrees of iris hypoplasia that are associated with additional ocular abnormalities. More than 90% of the causal mutations identified are found in the PAX6 gene, a transcription factor of critical importance in the process of neurogenesis and ocular development. Here, we investigate clinical, molecular, and craniofacial features of a large Brazilian family with congenital aniridia. Among the 56 eyes evaluated, phenotype variation encompassed bilateral total aniridia to mild iris defects with extensive variation between eyes of the same individual. PAX6 molecular screening indicated a heterozygous splice mutation (c.141 + 1G>A). Thus, we hypothesize that this splicing event may cause variation in the expression of the wild-type transcript, which may lead to the observed variation in phenotype. Affected individuals were more brachycephalic, even though their face height and cephalic circumference were not significantly different when compared to those of non-affected relatives. From this, we infer that the head shape of affected subjects may also be a result of the PAX6 splice-site mutation. Our data summarize the clinical variability associated with the ocular phenotype in a large family with aniridia, and help shed light on the role of PAX6 in neurocranial development.

Isolation, follicular density, and culture of preantral follicles of buffalo fetuses of different ages.

The aim of the present study was to determine the most desirable ovarian tissue section thickness to isolate preantral follicles (Experiment I), determine follicular density (follicles/mm(2) of cortex) of ovaries of fetal buffalo of different ages (Experiment II), and cultivate preantral follicles of buffalo fetuses (Experiment III). In Experiment I, ovary sections with different thicknesses (25, 50, 75, and 100 microm) had 415.0+/-285.2, 457.5+/-341.9, 585.0+/-309.3, and 685.0+/-278.8 isolated preantral follicles, respectively. In Experiment II, the follicular density of 46 buffalo fetuses with ages between 3 and 8 months was estimated to be between 0 and 7220, with means of 0.0, 2070.7+/-2190.3, 2570.8+/-1796.6, 2298.1+/-2286.5, 1277.5+/-1074.9, and 643.6+/-543.9 throughout the age range studied. The follicular density of 5-month-old fetuses was greatest, coinciding with the largest number of follicles isolated at this age. In Experiment III, preantral follicles isolated from the ovaries of buffalo fetuses aged from 5 to 9 months old were cultivated individually for 7 days in four different media: basic medium (Minimal Essential Medium (MEM), 10% SFB, kanamycin, pyruvate, glutamine, hypoxanthine) with additional ITS and FSH 0.5mg/ml (treatment 1); basic medium with FSH and EGF 100 ng/ml (treatment 2); basic medium with additional ITS, FSH, and EGF (treatment 3); basic medium supplemented with ITS and EGF (treatment 4). Integrity and morphological features, viability, and increase in diameter of follicles cultured in vitro were evaluated individually with an inverted microscope and an ocular micrometer. The results showed that follicle structure and form were maintained during culture. Growth and survival rates of treatments 1, 2, and 3 over 7 day culture were 23.25+/-17.06, 33.75+/-26.19, and 43.75+/-31.73 microm, and 31.3+/-22.7, 22.06+/-8.13, and 28.92+/-21.32%, respectively. However, neither growth nor survival was observed in treatment 4. In conclusion, this study showed that preantral follicles of buffalo fetuses can be cultured in vitro, and that FSH is essential for follicle survival.