Active immunization against GRF at 6 months of age delays puberty in beef heifers. The objectives of the present study were to determine whether active immunization against GRF at an earlier age would affect normal onset of puberty and follicular growth and to determine whether these changes were related to alterations in ovarian insulin-like growth factor I (IGF-I) or IGF binding protein (IG-FBP) messenger RNA (mRNA) levels. Heifers were immunized against human serum albumin (HSAi; n = 15) or against GRF conjugated to HSA (GRFi; n = 18) at 3 months of age. A third group of heifers was not immunized (CON; n = 16). Immunization against GRF delayed puberty beyond 13 months of age in 75% of treated heifers. Unilateral ovariectomy at 191 days of age revealed that the delay in puberty was associated with a reduction in the number of large ( > or = 7 mm in diameter) follicles. Large follicles were present in only 22% of GRFi heifers compared to 77% of HSAi heifers. The number of small ( < or = 3 mm in diameter) and medium (4 to 6 mm in diameter) follicles was not affected by GRFi. The percentage of 1- to 3-mm follicles that were atretic was not different between HSAi (65%) and GRFi (62%) heifers. Unilateral ovariectomy had no effect on age at puberty. Immunization against GRF decreased (P < 0.01) concentrations of IGF-I in serum (23 +/- 2 ng/ml) compared to HSAi heifers (109 +/- 11 ng/ml). IGF-I levels in follicular fluid (FFL) of medium and small follicles were also decreased by GRFi from 82 +/- 3 ng/ml in HSAi heifers to 48 +/- 6 ng/ml (P < 0.01). Levels of IGFBP-3 (determined by ligand blot analysis) in serum and FFL of small follicles were decreased by GRFi (P < 0.01). In contrast, IGFBP-2 serum levels were increased from 422 +/- 32 ng/ml in HSAi heifers to 657 +/- 6 ng/ml in GRFi heifers (P < 0.05). Likewise, IGFBP-2 levels in FFL from small and medium follicles were increased from 785 +/- 44 ng/ml to 926 +/- 44 ng/ml (P < 0.05). Ligand blot analysis indicated that IGFBP levels were lower in FFL from large vs. small follicles. The band intensities of IGFBP-4 and -5 were drastically reduced ( > 80%) while the decreases in IGFBP-2 and -3 were less marked ( < 50%). The decreased levels of IGFBP-5 in FFL from large follicles was not associated with an increase in proteolytic fragments detectable by immunoblot analysis. While mRNA transcripts for IGF-I, GH receptor, and IGFBP-2, -3, -4, and -5 were readily detectable in ovarian tissue, GRFi had no effect on ovarian levels of mRNA for each of these proteins. This suggests that the decrease in follicular development associated with GRFi may be related to changes in circulating IGF-I and/or IGFBPs.