To begin the process of identification and charactization of rat ovarian insulin-like growth factor binding proteins, we have undertaken to explore the ovarian expression, cellular localization, and hormonal regulation of the insulin-like growth factor binding protcin-2 (1GFBP-2) gene lor which an anligonadoiropic potential has recently been demonstrated. To this end, a solution hybridization/RNasc protection assay was employed wherein total ovarian RNA (2(Vg) from immature (21-23 davs old) female rats was hybridized with a 32P-laballed 1GFBP-2 nboprobe. As in liver, a single protected fragment (550 bases long.) corresponding to IGFBP-2 transcripts was identified in whole ovarian material. Cellular localization studies revealed the IGFBP-2 gene to be exclusively expressed in the ihcca-intcrstilial rather than the granulosa cell compartment. To confirm the cellular disinbution of the IGFBP-2 protein, media conditioned by cultured granulosa or theca-intcrsiitial cells weresubjectedtoimmunoprecipation using two IGFBP-2-directedpolyclonalanliscraExpectdly.bothantibodies(butnotnon-immunerabbitserum) readily.mmunoprccip.latcd the 28kDa rat IGFBP-2 species “cncratcd by hepatic BRL-3A cells. Similarly, both antibodies effectively immunoprccipitatcd an IGFBP the size ol rat IGFBP-2 elaborated by theca-micrstiiial cells. In contrast, neither antibody immunoprccipitatcd the 28-29kDa IGFBP species elaborated by granulosa cells otherwise readily apparent in conventional Western ligand blots. Hypophyscctomy resulted in a 3-fold decrease (P<0.05) in the relative (dcnsiiomctncally-quantidcd) abundance ol ovarian IGFBP-2 transcripts, a diametrically opposed effect (P<0.05) being noted at the level of the liver. In contrast, treatment ol immature hypophyscctomized rats with a dicthvlstilbcstrol-containing subcutaneous silastic implant for a total of 5 days resulted in a concordant 3-fold increase (P<0.05) in the relative abundance of IGFBP-2 transcripts in both ovary and liver when compared with untreated hypophyscctomized controls. Taken together, these findings document rat ovarian IGFPB-2 gene expression to be Ihcca-inlcrslitial (rather than granulosa) cell-selective, and subject to uprcgulatory control by pituitary principlc(s) and/or by estrogens. Although equally estrogen-dependent, hepatic IGFBP-2 gene expression proved constitutive in nature and subject to (diametrically opposed) inhibitory control by (potentially distinct) pituitary principlc(s).
ASJC Scopus subject areas