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Role and Gonadotrophic Regulation of X-Linked: MATERIALS AND METHODS(6)

Follicular Adenoviral Injection

To assess the role of XIAP in FSH-induced follicular development, XIAP content in the cultured follicles was manipulated by adenoviral XIAP antisense and sense (Myc-tagged) cDNA expression. After a 24-h culture in the absence of FSH in a 96-well plate, follicles were transferred onto a cell strainer (mesh size, 100 |xm; Becton Dickinson Labware, Franklin Lakes, NJ) in a 35-mm dish containing FCM. Replication-deficient adenovirus containing LacZ, XIAP sense, or XIAP antisense full-length cDNAs was injected into the follicles. ventolin inhaler

The volume of the virus injected was less than 10% (v/v) of the calculated follicular volume. Based on the follicular volume and estimated cell number, the amount of virus injected was multiplicity of infection (MOI) of 20 for XIAP antisense cDNA to assure adequate XIAP down-regulation. The MOI for the virus control (LacZ) was also 20. An MOI of three was chosen for XIAP sense cDNA, because it was sufficient to up-regulate XIAP expression. The FSH (5 ng/ml) was added to the cultures 24 h later, and the follicles were cultured for another 3 days. Preliminary studies indicated that follicles cultured with FSH at concentration of 100 ng/ml had higher XIAP level and were more resistant to XIAP down-regulation. In contrast, follicular XIAP level was low in the absence of FSH and less responsive to XIAP up-regulation by adenoviral sense expression. Thus, a lower concentration of oFSH (5 ng/ml) was used in subsequent cultures to achieve maximum response to XIAP up- and down-regulation by adenoviral cDNA expression.