Slot Blot Analysis
To corroborate the RT-PCR results, steady state amounts of FSHp mRNA were also measured by slot blot analysis as previously described. Amounts of mRNA for activin pB and follistatin were insufficient for detection by this method. Polyadenylated (poly[A]+) RNA was prepared from pituitary cells immediately following incubation and its integrity was confirmed by Northern blot analysis. Quantification of mRNA encoding the FSHp subunit was performed as described previously. Samples of poly(A)+ RNA (600 ng) were applied to nylon membranes in duplicate using a slot blot apparatus and cross-linked by exposure to ultraviolet radiation. Membranes were hybridized to radiolabeled cDNA for bovine FSHp. Membranes were exposed to film for 3 days, after which they were stripped by washing in boiling 0.1% SDS and probed with radiolabeled p-actin cDNA (188 base pair-amplified PCR product; see above). This procedure allowed for normalization of unequal loading among RNA samples on the nylon membrane. Autoradiographs were analyzed using the NIH 1.52 image analysis program. Concentrations of mRNA are expressed as percent of control values.
Concentrations of LH and FSH were assayed using previously described procedures. Reference preparations for LH and FSH were NIH-oLH-S24 and NIH-oFSH-S12, respectively. Mean limit of detection, intraassay coefficient of variation (CV), and interassay CV were 127 pg/ ml, 11.4%, and 12.2%, respectively, for the LH assay; and 6.0 ng/ml, 10.3%, and 10.8%, respectively, for the FSH assay.
Differences in media concentrations of gonadotropins and amounts of mRNA were determined by one-way AN-OVA using the general linear model procedure of SAS. Data obtained from multiple-culture wells were pooled for statistical analysis. When significant differences were found, means were compared by Tukey’s test. All quantitative data are presented as mean ± SEM.