IGF-1 stimulates synthesis of undersulfated proteoglycans and of hyaluronic acid by peritubular cells from immature rat testis.

The exposure of confluent peritubular (PT) cells from immature rat testis to insulin-like growth factor-1 (IGF-1) induced a time and dose-dependent increase of [35S]-sulfate and [3H]-D-glucosamine incorporations in newly synthesized proteoglycans (PG). This increased content of PG was the result of an enhancement of PG synthesis rather than a decreased rate of degradation. IGF-1 had no effect on the molecular weight of synthesized PG nor on the nature and distribution of the constitutive glycosaminoglycan chains, both in medium and in cell layer. The stimulation of PG synthesis by IGF-1 appeared to be due, at least partially, to an increase of glycosylation processes. IGF-1 effect was mediated by the classical tyrosine kinase signalling process, since IGF-1 action on PG synthesis was abolished by genistein and tyrphostin A9, two well known tyrosine kinase inhibitors. The increase of PG synthesis was accompanied with an undersulfation of constitutive glycosaminoglycan (GAG) chains (chondroitin sulfate and heparan sulfate chains) since the [35S]/[3H] ratio was reduced by about 20-25% in presence of IGF-1. Although the mechanism of hyaluronic acid synthesis was completely different from those of other GAG, IGF-1 also dramatically enhanced its production by PT cells.

Membrane associated proteoglycans in rat testicular peritubular cells.

Confluent testicular peritubular cells derived from immature rats were used to study membrane associated proteoglycans (PG). Peripheral material (heparin releasable), membrane and intracellular material (Triton X-100 releasable) were collected, purified by anion exchange chromatography then characterized by gel filtration and by hydrophobic interaction chromatography, followed by enzymatic digestion and chemical treatment. The peripheral material was constituted of two populations of PG (Kav = 0 and 0.10 on Superose 6 column), each containing both heparan sulfate proteoglycans (HSPG) and chondroitin proteoglycans (CSPG) and perhaps a hybrid PG (HSCSPG). These PG being not retained on an octyl Sepharose column, they were devoided of hydrophobic properties. The integral membrane proteoglycans isolated on the basis of their hydrophobic properties represented 20% of the Triton X-100 releasable material, and were exclusively constituted of proteoheparan sulfate. There were no relationships between this membrane HSPG and the peripheral HSPG as evidenced by pulse chase experiments. The mode of intercalation of the hydrophobic HSPG in the cell membrane was studied. The majority of these macromolecules (80%) were sensitive to trypsin and only a minor proportion (20%) were sensitive to phosphatidylinositol specific phospholipase C. Thus, about 80% of the hydrophobic HSPG were intercalated in the cell membrane by a hydrophobic segment of the core protein whereas about 20% were associated with the cell membrane via a phosphatidylinositol residue covalently bound to the core protein of the PG.

[Utilization of linoleate and alpha-linolenate in peritubular cells and Sertoli cells in culture]. / Utilisation du linoléate et de l'alpha-linolénate par les cellules péritubulaires et les cellules de Sertoli en culture.

The capacity of cultured peritubular cells to synthesize long-chain polyunsaturated fatty acids (PUFA) from the essential fatty acid (EFA) precursors 18:2n-6 and 18:3n-3 was tested, and compared to the PUFA biosynthesis in Sertoli cells. The concentrations of each EFA required to obtain maximal incorporation into membrane lipids were determined. The two EFA were added to the culture medium as free fatty acids complexed to albumin in a molar ratio of 12:1. When the substrates were added individually, the maximal levels of biosynthesis in peritubular cells were obtained with 0.70 microgram/ml of 18:2n-6 or 18:3n-3 in culture medium. With Sertoli cells, the concentration of 0.70 micrograms/ml linoleate or of 2.00 micrograms/ml alpha-linolenate in culture medium appeared to correspond to levels required for maximal incorporation and utilization of the n-6 PUFA or n-3 PUFA. Incorporation and metabolic utilization were always more important in cultured Sertoli cells than in cultured peritubular cells. The peritubular cell appeared to incorporate linoleate les efficiently than alpha-linolenate at identical concentrations. In agreement with observations in other cell systems (15), we found a preferential utilization of 18:3n-3 over 18:2n-6 by the delta 6 desaturase in the peritubular cell and in the Sertoli cell.