Development of a novel protocol for isolation and purification of human granulosa cells.

PURPOSE: To develop an optimal method of isolation and purification of human granulosa cells from ovarian follicular fluid. METHODS: Follicular fluid was collected from patients undergoing oocyte retrieval. A series of isolation and purification techniques was performed, involving density gradient centrifugation and use of different antibody-bead complexes. RESULTS: The highest percent yield of live purified granulosa cells came from density gradient centrifugation using sucrose polymer followed by positive selection of granulosa cells using primary antibody to MISRII and secondary antibody coupled to iron oxide beads. CONCLUSIONS: A novel protocol for granulosa cell purification has been developed yielding samples that are largely free of nondesirable cells. This protocol provides a purification solution, especially for patient samples that have significant RBC contamination.

Testosterone administration to elderly men increases skeletal muscle strength and protein synthesis.

Aging men develop a significant loss of muscle strength that occurs in conjunction with a decline in serum testosterone concentrations. We investigated the effects of testosterone administration to six healthy men [67 +/- 2 (SE) yr] on skeletal muscle protein synthesis, strength, and the intramuscular insulin-like growth factor I (IGF-I) system. Elderly men with serum testosterone concentrations of 480 ng/dl or less were given testosterone injections for 4 wk to produce serum concentrations equal to those of younger men. During testosterone administration muscle strength (isokinetic dynamometer) increased in both right and left hamstring and quadricep muscles as did the fractional synthetic rate of muscle protein (stable-isotope infusion). Ribonuclease protection assays done on total RNA from muscle showed that testosterone administration increased mRNA concentrations of IGF-I and decreased mRNA concentrations of insulin-like growth factor binding protein-4. We conclude that increasing testosterone concentrations in elderly men increases skeletal muscle protein synthesis and strength. This increase may be mediated by stimulation of the intramuscular IGF-I system.

Insulin-like growth factor-I increases expression of the porcine P-450 cholesterol side chain cleavage gene through a GC-rich domain.

The promoter/regulatory region of the porcine P-450 cholesterol side chain cleavage (P450scc) gene was cloned from a porcine genomic library. This gene contains a GC-rich region (-130-100) that mediates insulin-like growth factor I (IGF-I) and cAMP stimulation of gene expression in porcine granulosa cells. Stimulation of gene expression by cAMP occurs in 6 h, while IGF-I stimulation occurs in 24-48 h. This region is also responsive to insulin but not fibroblast growth factor. The effects of IGF-I and cAMP on gene expression in porcine granulosa cells are additive. In Y1 adrenal cells, the same region is responsive to cAMP but not to IGF-I. Gel shift assays using an oligonucleotide of this region and nuclear extract protein from porcine granulosa and Y1 adrenal cells identified three DNA-protein complexes (C1-C3). The binding activity of the complexes did not change with IGF-I or forskolin treatment of granulosa cells. Mutational analysis results were consistent with IGF-I regulating gene expression through the C2 DNA protein complex. Moreover, this complex binds differently in gel shift assay to mutant oligonucleotides with porcine and Y1 and nuclear extract protein. We conclude that IGF-I stimulates porcine P450scc gene expression through a distinct, cell-specific protein complex binding to a GC-rich domain.

Dexamethasone potentiates IGF-I actions in porcine granulosa cells.

To understand better interactions between glucocorticoids and insulin-like growth factor I (IGF-I) in the ovary, we studied the effects of dexamethasone on IGF-I stimulation of P-450 cholesterol side-chain cleavage enzyme (P-450scc) mRNA concentrations in porcine granulosa cells. Dexamethasone potentiated IGF-I-stimulated P-450scc mRNA concentrations and progesterone production in granulosa cell cultures. Time-course and dose-response studies showed that maximal enhancement occurred at a 1-microM dexamethasone concentration after 48 h of treatment. This potentiation was prevented by the glucocorticoid receptor antagonist RU-38486, 17 beta-hydroxy-11 beta-[-4-dimethyl-aminophenyl]estra-4,9,-dien-3-one (RU-486). We investigated mechanisms for this potentiation by performing IGF-I binding studies in porcine granulosa cells. Dexamethasone increased IGF-I binding, and Scatchard analysis showed this enhanced binding was caused by an increase in receptor concentration. Northern blot hybridization using a rat type I IGF-I receptor gene riboprobe showed that although dexamethasone alone did not increase IGF-I receptor mRNA concentrations, it did prevent a decrease in receptor mRNA concentrations caused by IGF-I. In addition, we used synthetic primers from conserved regions of the rat type I IGF-I receptor gene with total RNA from porcine granulosa cells and polymerase chain reaction to isolate a 615-base pair porcine type I IGF-I receptor cDNA clone. Ribonuclease protection assay results were similar to those found with the rat IGF-I receptor riboprobe. We conclude that dexamethasone potentiates IGF-I actions on steroidogenesis in the porcine ovary.