Gregory F. Erickson

A developmental study on the capacity of rabbit granulosa cells to respond to trophic hormones and secrete progesterone in vitro

To determine when undifferentiated rabbit granulosa cells first develop the capacity to secrete progesterone, pieces of intact ovaries from neonatal rabbits (newborn—30 days old) and pure granulosa cells from 150–1200 μm follicles at 60–600 days old were cultured in vitro for 6–10 days with human chorionic gonadotropin (HCG), Pergonal (LHFSH), dibutyryl cyslic AMP (Bu2CAMP), prostaglandin E2, estradiol-17β, and as controls. The culture medium was collected every 2 days, and progesterone, estrone, and estradiol-17β were measured by radioimmunoassay. None of the neonatal ovaries or granulosa cell cultures secreted estrone or estradiol-17β spontaneously or in response to stimulation by gonadotropins, Bu2CAMP, or prostaglandin E2. Control cultures of newborn and 7-day-old ovaries did not secrete progesterone, but ovaries from 17- and 30-day-old rabbits did. Gonadotropins and Bu2CAMP induced progesterone secretion in 7-day-old ovaries and stimulated its production 5-10-fold in ovaries at 17 and 30 days old, but prostaglandin E2 and estradiol-17β were without effect. Granulosa cells from all antral follicles (200–1200 μm) secreted progesterone spontaneously, and its production was stimulated 100–1000-fold with gonadotropins and Bu2CAMP, but not with estradiol-17β or prostaglandin E2. In contrast, granulosa cells from 100–150 μm preantral follicles from 200-day-old animals did not secrete progesterone under these culture conditions. These results demonstrate that rabbit granulosa cells differentiate the capacity to secrete progesterone at the time the primary follicle develops an antrum, and suggest the differentiation process involves the acquisition of the capacity to respond to gonadotropins perhaps by the synthesis or unmasking of gonadotropin receptors.

Evidence for a neutrophil-interleukin-8 system in human folliculogenesis.

OBJECTIVE This study was conducted to determine whether polymorphonuclear leukocytes (neutrophils) and the potent chemoattractant interleukin-8 are associated with follicle development in the normal human ovary. STUDY DESIGN We performed a morphometric analysis of neutrophils in 268 human ovarian follicles, of which 199 were preantral and 69 were antral. In each antral follicle the numbers of mitotic, apoptotic, and total granulosa cells were counted to determine healthy and atretic follicles. Interleukin-8 protein and messenger ribonucleic acid were detected by immunohistochemistry and in situ hybridization, respectively. RESULTS Antral follicles contained relatively large numbers of neutrophils within the theca vasculature. The density of neutrophil was twofold greater (p < 0.05) in atretic versus healthy follicles. The neutrophil index (neutrophils/granulosa cells x 1000) was inversely correlated to the number of granulosa cells per follicle. Immunoreactive interleukin-8 was detected in the theca and granulosa cells of most all antral follicles examined. Interleukin-8 messenger ribonucleic acid was demonstrated in theca and granulosa cells of some but not all follicles examined. CONCLUSIONS Neutrophils are present in the theca of developing antral follicles, increase in number during atresia, and are associated with expression of interleukin-8 in the follicle wall.

Prostaglandin F production in vitro by granulosa cells from rabbit pre-ovulatory follicles

Abstract The concentration of PGF in rabbit Graafian follicles increases at ovulation but the cell type responsible for PGF secretion has not been identified. We have found that a pure population of granulosa cells isolated from pre-ovulatory follicles of estrous rabbits secrete prostaglandin F in tissue culture (total secretion, 446 ng/10 days; 0.09 pg/cell/day). LH/FSH did not influence the rate of PGF secretion, but there was a 50% inhibition after dibutyryl cAMP treatment, and complete inhibition by indomethacin. These results indicate that granulosa cells could secrete the prostaglandin which accumulates in the follicle at ovulation, and that PGF secretion may be modified by the addition of cAMP to the medium.

PRODUCTION OF PROSTAGLANDIN F BY RABBIT GRANULOSA CELLS AND THECAL TISSUE

Prostaglandin production within the Graafian follicle may be of importance in relation to steroidogenesis and ovulation (see Lindner et al., 1974). In culture, human and rabbit follicles synthesize prostaglandin (PG) F, and this capacity can be stimulated by exogenous gonadotrophins (Plunkett, Moon, Zamecnik & Armstrong, 1975; Moon, Zamecnik & Armstrong, 1974). However, the cell type responsible for this prostaglandin production has not been established. Previous studies showed that granulosa cells from preovulatory follicles of the rabbit secreted PGF when cultured as a monolayer for 10 days (Challis, Erickson & Ryan, 1974), but the activity of thecal tissue was not examined. In the present study we have compared the capacity of granulosa and thecal cells to produce PGF in tissue culture, and have observed the effects on this capacity of exogenous gonadotrophin, arachidonic acid and oestradiol. Tissue was obtained from non-pregnant sexually mature New Zealand White rabbits (2-0-3 · 5 kg). The animals were killed by cervical dislocation, and the ovaries removed and placed at 4°C in McCoys 5a culture medium (Erickson, Challis & Ryan, 1974). Granulosa cells were isolated and cultured in 5 ml McCoys 5a medium (modified) for 2 days in a humidified 95 % air/5 % C02 incubator at 37°C. The details of this procedure and the composition of the medium have been published previously (Erickson et al., 1974). Each culture contained the granulosa cells from 4 preovulatory Graafian follicles (1000-1200 μ diameter). Thecal tissue from 4 preovulatory follicles was cultured