Inhibitory action of porcine follicular fluid upon granulosa cell luteinization in vitro: assay and influence of follicular maturation.

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.

Follicular fluid stimulation of steroidogenesis in immature granulosa cells in vitro.

Granulosa cells from small (1-2 mm) immature porcine follicles were cultured in monolayer in culture media composed of equal parts of culture medium 199 and either (a) fluid from small follicles, (b) fluid from large (6-12 mm) follicles or (c) adult female porcine serum for 6 days, with or without 100 ng LH and/or 2 microgram FSH/ml. Both basal and gonadotrophin-stimulated progesterone secretion were greater in the presence of fluid from large follicles than in serum, for all 6 days. After 4 days of culture, fluid from small follicles enhanced gonadotrophin-stimulated progesterone secretion over that occurring in serum, but to a lesser extent than fluid from large follicles. These studies suggest the presence of a maturation stimulating molecule(s) in follicular fluid which increases in activity or concentration as the follicles enlarge. This factor may be essential for normal granulosa cell maturation in vivo.

Follicular fluid depression of ovarian granulosa cell adenylyl cyclase activity.

Previous studies have demonstrated that fluid from small (1--2 mm) porcine follicles is inhibitory of LH stimulation of cyclic AMP accumulation, progesterone secretion and morphological luteinization of granulosa cells from large (6--12 mm) porcine follicles. The present study measured adenylyl cyclase in granulosa cells following 2-day incubations in either the inhibitory culture medium containing 50% fluid from small follicles or in luteinization permissive media: 50% fluid from large follicles or 50% porcine serum. Basal, LH, epinephrine and NaF stimulation of adenylyl cyclase activity were all significantly reduced in the cells incubated with fluid from small follicles as compared to cells incubated in the control media. The data presented in this paper, in conjunction with previously publised data suggest that the inhibitory fluid from small follicles, alters both receptor--cyclase coordination and adenylyl cyclase activity.

The influence of follicular fluid on progesterone secretion by porcine granulosa cells in vitro.

Porcine follicular fluid contains at least two factors which can modify porcine granulosa cell function in vitro. One factor, found primarily in fluid from 1--2 mm follicles inhibits LH stimulated cyclic AMP accumulation, basal and LH stimulated progesterone secretion, and morphological luteinization of granulosa cells from medium sized (3--5 mm) and large (6--12 mm) follicles. Following inhibition by the follicular fluid luteinization inhibitor, incubation with both OH and a phosphodiesterase inhibitor partially restores cyclic AMP accumulation and fully restores progesterone secretion. This suggests that the defect induced by the luteinization inhibitor is primarily an alteration of cyclic nucleotide metabolism. Another factor found primarily in fluid from large preovulatory follicles stimulates basal and gonadotrophin stimulated progesterone secretion by granulosa cells from small antral follicles. We hypothesize that it may be necessary for the fluid within an individual follicle to shift from being inhibitory of luteinization to being permissive or stimulatory for the granulosa cells to mature rather than become atretic.

Seasonal variation in porcine granulosa cell responsiveness to stimulation of progesterone secretion in vitro.

Ten years of data were analyzed for an effect of season on basal and stimulated progesterone secretion by porcine granulosa cells in vitro. Basal progesterone secretion by granulosa cells from large preovulatory follicles exhibited a marked depression during the summer months, as compared to the rest of the year. The ability of granulosa cells from small (1-2 mm) or medium (3-5 mm) follicles to respond to stimulation of progesterone secretion by either follicle-stimulating hormone (FSH) or follicular fluid was also markedly depressed during the summer months. These observations on decreased steroidogenic capacity of porcine granulosa cells in vitro during the summer parallel observations of decreased reproductive function of pigs in vivo. They suggest that the decreased reproductive capacity of pigs in summer may be due, in part, to a decreased capacity for follicular development within the ovary as a result of an altered neuroendocrine signal rather than due only to an altered neuroendocrine signal.

The actions of follicular fluid factors on steroidogenesis by cultured ovarian granulosa cells.

Fluid from porcine follicles alters estrogen and progesterone secretion by porcine granulosa cells in vitro. The specific actions of the follicular fluids depend on the size of the follicles from which both the fluid and the cells are obtained. Fluid from large, primarily preovulatory follicles enhances both basal estrogen secretion and basal and hormonally stimulated progesterone secretion by cells from small follicles. Estrogen secretion by cells from larger follicles is also enhanced but fluid from large follicles is only equivalent to serum in supporting progesterone secretion by these more mature cells. Fluid from small follicles exhibited weak stimulatory actions on cells from small follicles but inhibited basal and hormonally stimulated progesterone secretion by cells from larger follicles. A hypothetical model is proposed for a physiological role for the stimulatory and inhibitory follicular fluid factors. Only those small follicles containing the stimulator become sufficiently responsive to gonadotrophins and produce adequate estrogen to allow further follicle maturation. The remaining small follicles possess the inhibitor which blocks responsiveness to gonadotrophins, and contributes to atresia. Many questions remain concerning the identity of the factors, which cells produce them and how their production is controlled.

Follicular fluid stimulation of progesterone secretion: time course, dose-response and effect of inhibiting de novo cholesterol synthesis.

This study extends our previous observations of follicular fluid stimulation of progesterone secretion by granulosa cells from immature porcine antral follicles in vitro. Granulosa cells were incubated with either charcoal-treated fluid from large porcine follicles or serum in TC medium 199. Within minutes of the addition of follicular fluid to the cells, enhanced progesterone secretion was observed. A second, more substantial enhancement of progesterone secretion was observed after an additional incubation of 24 to 48 h. As we were unable to detect significant amounts of progesterone within either freshly collected or incubated cells, follicular fluid appears to stimulate both the synthesis and secretion of progesterone rather than just the secretion of preformed progesterone. Inhibition of de novo cholesterol synthesis with an inhibitor of the conversion of 7-dehydrocholesterol to cholesterol, reduced progesterone secretion by cells incubated with serum to a greater extent than by cells incubated with follicular fluid. These studies suggest that follicular fluid may stimulate both de novo cholesterol synthesis and progesterone synthesis from preformed cholesterol. A dose-response test for follicular fluid stimulation of progesterone secretion indicated that 30% follicular fluid in TC medium 199 was as stimulatory as 50% follicular fluid. Ten percent follicular fluid was not stimulatory by itself, but in combination with 1 microgram of FSH/ml (ovine follicle-stimulating hormone), stimulated progesterone secretion to a greater extent than 30% or 50% follicular fluid alone.

Morphometric analysis of in vivo development of porcine ovarian granulosa cells in preovulatory antral follicles.

Granulosa cells (GC) from immature (1-2 mm), developing (3-5 mm), and preovulatory (6-12 mm) antral porcine follicles were examined by stereological, ultrastructural techniques. Partial cell volumes occupied by nuclei or mitochondria did not differ significantly as follicles enlarged. Whorled smooth endoplasmic reticulum (ER) increased significantly in large follicles compared to either small or medium sized follicles. Whorled rough ER elements were present in similar amounts in small and medium sized follicles and absent in large follicles. The GC of large follicles contained significantly more lipid and Golgi complexes than that of small follicles, but the lipid and Golgi complex content of GC from medium follicles was not significantly different from that of either small or large follicles. Proportions of total cell volumes occupied by lysosomes and multivesicular bodies increased as follicular size increased. This confirms earlier qualitative studies, and provides a quantitative in situ basis for future in vitro studies.

The glycosaminoglycan chondroitin-4-sulfate alters progesterone secretion by porcine granulosa cells.

Porcine granulosa cells from small (1-2 mm), medium (3-5 mm), and large (6-12 mm) antral follicles were cultured in monolayer for 2 to 3 days with 0 to 3 mg of chondroitin-4-sulfate (C-4-S)/ml in the presence or absence of 0.5 microgram follicle-stimulating hormone (NIH-FSH-S13)/ml. Testosterone (1.4 microgram/ml) was added to some cultures as substrate for estrogen synthesis. Progesterone and estrogen secreted into the media were measured by radioimmunoassay. Concentrations of C-4-S similar to concentrations of chondroitin sulfates (CS) reported for small antral or atretic follicles inhibited both basal and FSH-stimulated progesterone secretion. Progesterone secretion was not inhibited by C-4-S when pregnenolone was added to the media. Thus 3 beta-hydroxysteroid dehydrogenase activity was not inhibited by C-4-S. Estrogen secretion was also not inhibited by even the highest concentration of C-4-S tested. Testosterone did not influence C-4-S inhibition of progesterone secretion. Granulosa cells from medium-sized follicles were more sensitive to C-4-S than cells from small follicles. Granulosa cells from large follicles were completely resistant to C-4-S inhibition of progesterone secretion. These observations suggest that C-4-S may play a role in altering gonadotrophin-stimulated and basal progesterone secretion in follicles during differentiation of granulosa cells.

Follicular fluid effects on progesterone secretion are not due to follicle-stimulating hormone or steroids.

An antiserum raised against porcine follicle-stimulating hormone (FSH) was unable to eliminate the stimulatory action of fluid from large antral porcine follicles on progesterone secretion by granulosa cells from small antral porcine follicles. The same titers of the antiserum were completely effective at eliminating the effect of 2 micrograms of NIH-FSH-P12, whereas maximal stimulation of progesterone secretion was observed with 0.5 micrograms FSH/ml. The androgen and estrogen concentrations measured in charcoal-treated inhibitory follicular fluid from small porcine antral follicles and from stimulatory follicular fluid from large follicles were added separately and together to culture media supplemented with serum to determine if these low concentrations (5 X 10(-11) to 5 X 10(-10) M) of steroids could mimic the actions of follicular fluid on progesterone secretion. Neither the inhibitory nor the stimulatory actions of the follicular fluids could be mimicked by these low concentrations of steroids. Higher concentration of steroids (10(-8) to 10(-7) M range) did stimulate progesterone secretion as reported by others. Our data indicate that the actions of charcoal-treated follicular fluids on granulosa cell progesterone secretion cannot be explained by difference in FSH or steroid contents between the inhibitory and stimulatory fluids and serum.

Morphological correlates of follicular fluid stimulation of steroidogenesis in immature porcine granulosa cells.

Factors in porcine ovarian follicular fluid are known to influence steroidogenesis in cultured ovarian granulosa cells. This study examined whether ultrastructural changes characteristic of normal maturation and/or atresia accompany the steroidogenic alterations. Two and 5 day incubations of immature porcine granulosa cells were performed in media supplemented with either serum or follicular fluids (FFL) from mature follicles. Under these conditions both oestrogen and progesterone secretion were stimulated in FFL supplemented cultures as compared to serum supplemented cultures. Cells in serum exhibited increased size, number and volume of lysosomes and resembled in vivo atretic cells. In comparison, the FFL treated cells had greatly increased steroid output, numerous microvilli and increased size, number and volume of electron dense lipid droplets after 2 days of culture although the differences declined by day 5 of culture. This suggests that mature FFL contains a factor(s) stimulating granulosa maturation while inhibiting ultrastructural correlates of follicular atresia.