Stimulation of fibronectin production and deposition by chicken granulosa cells in vitro by epidermal growth factor and transforming growth factor alpha.

Experiments were conducted in vitro to examine the effect of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) on the production of fibronectin (deposited, secreted into medium and cell-associated) by hen granulosa cells isolated from the largest (F1; about 35 mm in diameter, mature) and third largest (F3; 15-20 mm in diameter) preovulatory follicles, as well as from a pool of immature small yellow follicles (6-8 mm in diameter). The cells were incubated in culture wells coated with type IV collagen or in wells without collagen coating, and the amounts of fibronectin produced were measured using a specific ELISA. The total amount of fibronectin produced by unstimulated cells was greatest in wells containing F1 cells and increased with time. The amount of fibronectin deposited by unstimulated cells was greatest in wells containing F1 cells and was much higher in collagen-coated wells than in uncoated wells. Both EGF and TGF-alpha increased the quantity of fibronectin deposited by granulosa cells in collagen-coated and uncoated wells. Fibronectin secreted into the medium by unstimulated cells also increased with the stage of follicular maturation and was enhanced by EGF and TGF-alpha. The quantity of cell-associated fibronectin in granulosa cells in collagen-coated and uncoated wells was also increased by these growth factors. Type IV collagen did not have any appreciable effect on the amount of fibronectin present in the incubation medium or on cell-associated fibronectin. Because of its marked effect on deposited fibronectin, there were greater total quantities of fibronectin in culture wells coated with type IV collagen. These results demonstrate that EGF and TGF-alpha stimulate the production and deposition of fibronectin by chicken granulosa cells. The results also suggest that in combination with collagen IV, these growth factors can regulate the formation of the extracellular matrix (for example, basal lamina) of the hen ovarian follicle.

Progesterone stimulates fibronectin production by chicken granulosa cells in vitro.

Experiments were conducted in vitro to examine the effect of progesterone on fibronectin production by chicken ovarian granulosa cells. Granulosa cells isolated from the largest (F1; mature) and third-largest (F3; developing) preovulatory follicles as well as form a pool of immature small yellow follicles (SYF) of the domestic chicken ovary were incubated in serum-free Medium-199 and the amounts of fibronectin and progesterone produced were quantified by enzyme-linked immunosorbent assay and radioimmunoassay respectively. The amounts of basal fibronectin and progesterone produced by granulosa cells from F1, F3 and SYF follicles increased with advancing stages of follicular development. Thus, the quantity of basal fibronectin secreted by granulosa cells was directly proportional to the amount of progesterone produced by them. Exogenously supplied progesterone increased the amount of fibronectin secreted by F1 and F3 cells in a dose-dependent manner, but its effect on SYF cells was marginal. Cyanoketone (an inhibitor of progesterone synthesis) suppressed basal fibronectin production by F1 and F3 granulosa cells and its inhibitory action was reversed by exogenous progesterone. The progesterone antagonist RU 486 also attenuated basal fibronectin production by F1 and F3 granulosa cells, but only the highest concentration affected SYF cells. The inhibitory effect of RU 486 was diminished in the presence of exogenous progesterone. These data show that progesterone regulates fibronectin production by chicken granulosa cells. They suggest that in avian granulosa cells, endogenous progesterone can stimulate fibronectin synthesis in an intracrine or autocrine manner.

Chicken gonadotropin-releasing hormones enhance soluble and insoluble fibronectin production by granulosa cells of the domestic fowl in vitro.

Experiments were conducted in vitro to examine the effect of two chicken gonadotropin-releasing hormones, cGnRH-I ([Gln8]-GnRH) and cGnRH-II ([His5,Trp7,Tyr8]-GnRH), on fibronectin (soluble and insoluble) production by chicken granulosa cells isolated from the largest (F1; about 35 mm in diameter), and third largest (F3; 15 to 20 mm in diameter) preovulatory follicles as well as from a pool of immature small yellow follicles (SYF; 6 to 8 mm in diameter). The amounts of soluble fibronectin (fibronectin secreted into the incubation medium) and insoluble fibronectin (fibronectin associated with cells plus fibronectin attached to culture substratum) were quantified with a specific ELISA. Fibronectin secreted into the incubation medium (soluble fibronectin) by unstimulated cells increased with advanced stages of follicular maturation. Addition of both cGnRH-I and -II increased the amount of fibronectin secreted into the incubation medium by all follicular cell types. The amount of insoluble fibronectin in culture wells that contained unstimulated cells also increased with advanced stages of follicle development. Both cGnRH-I and -II increased the quantity of insoluble fibronectin by granulosa cells from all follicle types. Total (soluble plus insoluble) fibronectin production was elevated when cGnRH-I or -II was added to F1, F3, and SYF granulosa cells. The magnitude of cGnRH-I or -II stimulation (percentage increase) of soluble, insoluble, or total fibronectin production was calculated as a multiple of the unstimulated (control) value for each follicle type, and they were greatest in cells derived from developing and immature follicles. These results indicate that homologous cGnRH-I and -II are capable of directly modulating the physiology of the avian ovary.

Influence of follicular maturation on the deposition of fibronectin by chicken granulosa cells in vitro--effect of 8-bromo cyclic AMP.

Fibronectin deposition by avian granulosa cells and the effect of 8-bromo cAMP (8-br-cAMP) on this process was investigated in vitro. Granulosa cells obtained from the first (F1) and third (F3) largest preovulatory follicles and from small yellow follicles (SYF) were incubated in serum free medium 199 for 12 or 24 hr with and without 8-br-cAMP. Fibronectin deposited in incubation wells secreted in medium or associated with cells was measured by ELISA. Basal fibronectin deposition increased with time and was greatest for F1 cells. Within 12 hr, 8-br-cAMP enhanced fibronectin deposition dose-dependently by F3 and SYF cells, but not by F1 cells. The amount of fibronectin deposition caused by 8-br-cAMP (which was greater after 24 hr incubation) was 0.10- to 0.16-fold for F1 cells; 0.20- to 0.81-fold for F3 cells and more than 30-fold for SYF cells. Fibronectin secreted in the medium by unstimulated cells was also greatest in F1 cells. 8-bromo cAMP stimulated fibronectin secretion in medium dose-dependently by F3 and SYF cells, however, it had only a marginal stimulatory effect on this process in F1 cells. Cell-associated fibronectin was not increased significantly by 8-br-cAMP in F1, F3 or SYF cells. Total (deposited plus medium plus cell associated) fibronectin production was elevated in dose- and time-dependent manner by 8-br-cAMP in F3 and SYF cells, but only at high concentrations in F1 cells. Like deposited fibronectin, the relative effect of 8-br-cAMP on fibronectin secreted in the medium and on total fibronectin production was greatest in immature SYF cells. These results demonstrate that fibronectin synthesized and deposited by unstimulated chicken granulosa cells in vitro increase concomitantly with follicular maturation and cellular differentiation, and the relative change in fibronectin production caused by 8-br-cAMP was greatest in immature SYF cells.

Follicle-stimulating hormone-enhanced fibronectin production by chicken granulosa cells is influenced by follicular development.

Experiments were conducted in vitro to examine the effect of ovine follicle-stimulating hormone (FSH) on various fractions of fibronectin (deposited, secreted in medium, and cell-associated) resulting from culture (12 or 24 h) of chicken granulosa cells isolated from the largest (F1) and third largest (F3) preovulatory follicles as well as from a pool of immature small yellow follicles (SYF). Fibronectin in each fraction was quantified with a specific ELISA. The amount of fibronectin deposited in culture wells containing unstimulated cells increased with time and was greatest in wells containing F1 cells. Follicle-stimulating hormone increased the quantity of fibronectin deposited within 12 h by F3 and SYF cells, but not by F1 cells. The magnitude of FSH-enhanced fibronectin deposition was greatest in cells derived from immature SYF. Fibronectin secreted in the medium by unstimulated cells also increased with the stage of follicular maturation. Follicle-stimulating hormone increased the amount of fibronectin secreted in the medium by F3 and SYF cells. The quantity of fibronectin associated with cells was increased by FSH in all cell types. Total (deposited plus medium plus cell-associated) fibronectin production was elevated in a dose- and time-dependent manner when FSH was added to F3 and SYF granulosa cells, but the gonadotropin was without effect in F1 cells. The magnitude of FSH stimulation (fold increase) of total fibronectin production was calculated as a multiple of the unstimulated (control) value for each cell type. The relative change in total fibronectin production resulting from addition of FSH (after 24 h incubation) was .02- to .24-fold in F1 cells as compared with .33- to .92-fold in F3 cells and 2.75- to 4.38-fold in SYF cells. These results indicate that FSH stimulates fibronectin production by chicken granulosa cells, and this stimulatory effect decreases as the follicle matures.

Plasma progesterone, luteinizing hormone concentrations, and granulosa cell responsiveness in heat-stressed hens.

Plasma progesterone and luteinizing hormone (LH) profiles were obtained during the first ovulatory cycle of heat-stressed (HS, 35 C; n = 24) and unstressed (US, 17 to 27 C; n = 24) hens using 30-min sampling intervals beginning approximately 6 h prior to ovulation. Progesterone levels from HS hens were lower from 6 h [.07 +/- .01 (SE) versus 1.66 +/- .25 ng/mL; P = .008] to predicted ovulation (.06 +/- .006 versus .70 +/- .18 ng/mL; P = .07). Likewise, LH levels from HS hens were lower from 6 h (1.55 +/- .16 versus 3.86 +/- .34 ng/mL; P = .007) to predicted ovulation (1.63 +/- .18 versus 2.50 +/- .27 ng/mL; P = .01). Eggs from HS hens were more often laid early (less than 24 h) than eggs from US hens (71.42 versus 13.33%, respectively; P = .01), but US hens more often laid eggs of a normal oviposition interval length (24 to 26 h) compared with HS hens (73.34 versus 14.29%; P = .0005). The percentage of delayed eggs (greater than 26 h) was not different (US, 14.29 versus HS, 13.37%; P = .75) between the two treatment groups. Basal production of progesterone by dispersed granulosa cells from US hens was 97.62 +/- 16.01 ng/mL. Challenge by LH increased this to 417.50 +/- 53.38 ng/mL (P = .0001). In contrast, basal progesterone secretion by cells from HS hens was 40.25 +/- 6.60 ng/mL (P = .0001) and LH challenge failed to increase progesterone production.(ABSTRACT TRUNCATED AT 250 WORDS)