Inhibitory Action of Porcine Follicular Fluid Upon Granulosa Cell Luteinization in Vitro: Assay and Influence of Follicular Maturation

Abstract

Culture medium 199 supplemented with follicular fluid from 1-2 mm antral porcine follicles inhibited spontaneous luteinization of granulosa cells from preovulatory porcine follicles in vitro. Three characteristics of luteinization were inhibited: morphological transformation, progesterone secretion, and accumulation of cyclic AMP in response to LH. The last was inhibited more effectively by culture media containing 50% follicular fluid than by media containing 20% follicular fluid. The inhibitory actions of the follicular fluid were not altered by charcoal or petroleum ether extraction. Follicular fluid from large follicles (6-12 mm) did not exhibit any of these inhibitory actions. These observations may indicate the presence of a luteinization inhibitor in the fluid of small follicles which (1) is lost by the time the follicle reaches the preovulatory stage, or (2) is overcome by a stimulatory agent which may accumulate as the follicle grows.

Evidence for ovarian “inhibin”: Suppression of the secondary rise in serum follicle stimulating hormone levels in proestrous rats by injection of porcine follicular fluid

“…For porcine serum, porcine follicular fluid1, and porcine follicular fluid2, estradiol = 28, 103, and 192 pg/ml, respectively; progesterone = 0.16, 1.9, and 3.2 ng/ml, respectively; and testosterone = 36, 950, and 450 pg/ml, respectively. These concentrations represented at least a 1000-fold reduction compared to non-charcoal-extracted fluid (7,18,19). The three biological fluids were also tested for FSH-like activity Abbreviations: LH, luteinizing hormone (lutropin); FSH, follicle stimulating hormone (follitropin).…”

“…Porcine follicular fluid was collected in two batches (PFF1 and PFF2) from 3-to 10-mm follicles of ovaries collected from a nearby meat packing plant and charcoal extracted for removal of steroids, as described previously (17,18). Porcine serum was obtained from Grand Island Biological Co. and was similarly treated for use as a control injection.…”

Blockade of recruitment of ovarian follicles by suppression of the secondary surge of follicle-stimulating hormone with porcine follicular field.

“…The same phenomenon has been seen in another group of similarly treated rats (unpublished observations). This progesterone suppression may result from an action, on the ovary, of a "luteinization inhibitor" found in PFF (16).…”

Activin A reduces luteinisation of human luteinised granulosa cells and has opposing effects to human chorionic gonadotropin in vitro

“…It is therefore believed that luteinisation is a differentiation pathway that is programmed before antral formation, and the only way the follicles can escape this fate is by inhibitory factors (Wehrenberg & Rune 2000). Literature suggests that the follicle itself can provide a milieu in order to discourage luteinisation (Channing et al 1980, Eppig et al 1997, Murphy 2000, and inhibitor(s) of such magnitude may actually be present in follicular fluid (Ledwitz-Rigby et al 1977, Channing et al 1978 or come directly from the oocyte itself (Vanderhyden & Macdonald 1998, Brankin et al 2003. It therefore appears that the LH surge is able to remove such inhibitory factors, disrupt connections between granulosa cells and the oocyte and/or induce genes that facilitate luteinisation.…”