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)

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.

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.

Asem EK, Conkright MD, Novero RP. Progesterone stimulates fibronectin production by chicken granulosa cells in vitro. Eur J Endocrinol 1994;130:159–65. ISSN 0804–4643 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 from 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. EK Asem, Department of Physiology and Pharmacology, School of Veterinary Medicine, Purdue University, 1246 Lynn Hall, West Lafayette, IN 47907-1246. USA

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.