Interleukin-2 affects steroidogenesis and numbers of bovine ovarian granulosa cells but not thecal cellsin vitro

The effect of recombinant bovine interleukin-2 (IL-2) on steroidogenesis and numbers of bovine ovarian granulosa and thecal cells has been studied. Granulosa cells have been examined from both small (surface diameter ≤5 mm) and large (≥8 mm) follicles, whereas thecal cells from only large follicles were utilized. Estradiol and progesterone production per cell by granulosa cells from large follicles was 2- to 3-times greater than those from small follicles. Increasing doses of IL-2 significantly attenuated FSH-induced estradiol production by cells from small follicles but not large follicles. In general, progesterone production per cell by granulosa cells was almost double that of thecal cells. Moreover, IL-2 significantly attenuated FSH-induced progesterone production by granulosa cells from small and large follicles but had no effect on LH-induced progesterone or and-rostenedione production by thecal cells. Co-treatment of TNFα with IL-2 enhanced the responsiveness of granulosa cells to IL-2. The effect of IL-2 on the numbers of granulosa and thecal cells were studied independently under serum-free conditions and media enriched with 10% fetal calf serum. In serum-free medium containing insulin, IL-2 dosage significantly increased numbers of granulosa cells from large follicles, whereas IL-2 had no effect on numbers of granulosa cells from small follicles or thecal cells from large follicles. When cells were grown in medium enriched with serum, increasing doses of IL-2 significantly inhibited numbers without affecting viability of granulosa cells from small follicles, but had no effect on numbers of thecal cells. Thus, it appears that granulosa cells are more sensitive to IL-2 than are thecal cells. Approved for publication by the Director, Oklahoma Agriculture Experiment Station. This research was supported in part under project H-2088.     

Influence of cortisol on insulin- and insulin-like growth factor 1 (IGF-1)-induced steroid production and on IGF-1 receptors in cultured bovine granulosa cells and thecal cells

During stress, hyperactivity of the adrenal gland can directly and indirectly inhibit ovarian function. However, little evidence existed to support the notion that glucocorticoids could influence insulin-like growth factor 1 (IGF-1) action within the ovary. Therefore, the effect of cortisol on IGF-1-induced granulosa and thecal cell function was evaluated. Granulosa and thecal cells from bovine ovarian follicles were cultured for 2 d in the presence of 10% fetal calf serum and then cultured for an additional 2 d in serum-free medium with added hormones. Cortisol had little or no effect (p>0.05) on IGF-1-induced progesterone production by granulosa cells from both small (1–5 mm) or large (≥8 mm) follicles. Also, cortisol had little or no effect (p>0.05) on basal, insulin-, or IGF-1-induced estradiol production by granulosa cells from small or large follicles, or on the number of IGF-1 receptors in granulosa cells from small follicles. Cortisol had no effect (p>0.10) on insulin-induced granulosa cell numbers, but increased IGF-1-induced granulosa cell numbers. In thecal cells, doses of 1–100 ng/mL of cortisol increased (p<0.05) insulin- and IGF-1-induced thecal cell numbers by 10–20%, progesterone production by 18–36%, and androstenedione production by two- to fourfold. The estimated dose of cortisol necessary to stimulate 50% of the maximum androstenedione production in the presence of IGF-1 was 7 ng/mL. In contrast, cortisol decreased (p<0.05) the number of IGF-1 receptors in thecal cells by 45%. In conclusion, cortisol at physiological levels can directly influence ovarian follicular function in cattle, especially thecal androstenedione production.     

Tumor necrosis factor-α (TNF-α) inhibits steroidogenesis of bovine ovarian granulosa and thecal cells in vitro

The effect of recombinant bovine tumor necrosis factor-α (TNF-α) on steroidogenesis and numbers of bovine ovarian granulosa and thecal cells has been studied, and specific binding sites for ¹²⁵I-TNF-α on ovarian cells have been determined. Granulosa cells have been examined from small (surface diameter 1–5 mm) follicles, whereas thecal cells from large (≥ 8 mm) follicles were utilized. Increasing doses of TNF-α significantly attenuated insulin- and IGF-I-induced estradiol production by granulosa cells from small follicles, but had no effect on basal estradiol production. Moreover, TNF-α significantly attenuated insulin- and LH-induced androstenedione production by thecal cells from large follicles. TNF-α had little or no effect on the numbers of granulosa and thecal cells in these same studies. Specific high-affinity, low-capacity binding of ¹²⁵I-TNF-α was also demonstrable in granulosa and thecal cells. Thus, it appears that TNF-α inhibits insulin-and IGF-I-induced estradiol production by granulosa cells and androstenedione production by thecal cells via TNF-α binding to its own receptor.     

Specific binding of [3H]heparin to bovine granulosa cell membranes

Bovine granulosa cell membranes from small (SFM) and large (LFM) antral follicles were incubated with [3H]heparin, a commercial radioactively labeled glycosaminoglycan (GAG). Binding was specific, reversible, saturable, and dependent on time, pH, ionic strength and divalent cations. SFM exhibited different [3H]heparin binding characteristics compared to LFM. The addition of a physiological concentration of calcium (2 mM) yielded significant differences (P less than 0.02) in [3H]heparin binding for SFM (87 590 +/- 4206 dpm/10(6) cells) compared to LFM (55 230 +/- 2816 dpm/10(6) cells). SFM and LFM showed maximum [3H]heparin binding at pH 6.5 and pH 5.5, respectively. Increasing the ionic strength by addition of 0.07-2.0 M NaCl interfered with binding. Addition of unlabeled heparin (0.1-100 micrograms/ml) displaced [3H]heparin bound to SFM and LFM in a dose-dependent manner, as did dextran sulfate, a non-GAG sulfated branched polysaccharide. Commercial chondroitin sulfate ABC displaced the bound [3H]heparin only at doses between 50 and 500 mg/ml. GAGs purified from FF suppressed binding 39% at a concentration of 5.9 mg/ml. Photomicrographs of fluorescein-labeled heparin bound to granulosa cells showed localized areas of heparin binding to the cell surface. These experiments demonstrated that the GAG heparin specifically bound to bovine granulosa cell membranes, and that significant differences existed between the binding characteristics of SFM and LFM.