Differential steroid and gonadotropin response by individual tertiary porcine follicles in vitro. Possible physiologic role of atretic follicles

Since atretic follicles contain significant amounts of androgen and/or progesterone in their follicular fluid, we examined whether they also contribute to ovarian steroid secretion. Steroid secretion by atretic porcine follicles and their responsiveness to FSH was assessed by a perifusion system that allows for separate dynamic incubation of whole follicles in vitro. Identically treated nonatretic follicles of comparable size served as a reference group. The extent of granulosal pyknosis, on which the staging of atresia was based, was inversely related to follicular estradiol (E2) secretion and its responsiveness to FSH. Both basal and FSH-stimulated secretion of testosterone (T), androstenedione (A), and progesterone (P) were maintained by follicles in all stages of atresia. Secretion of A by late atretic follicles was greater than that in earlier stages or by nonatretic follicles. Atretic follicles may therefore release comparable or larger amounts of androgen and P into their intraovarian environment than do nonatretic follicles. We examined whether steroids secreted by atretic follicles in vitro could be utilized by nonatretic follicles. A static incubation system was used that allows for simultaneous incubation of a number of individual follicles. When nonatretic follicles were exposed to A, T, or P in physiologic concentrations (10(-7)-10(-5) M), their secretion of E2 increased 2-8-fold. Doses of FSH or LH that stimulated follicular steroid in vitro had no additional stimulatory effect when combined with A or P treatment, respectively. In conclusion, atretic follicles may contribute significantly to intraovarian levels of androgen and P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regeneration of atretic sheep ovarian follicles in vitro.

Large (4--6 mm diam.) and small (2--3 mm) atretic follicles were removed from sheep ovaries during the luteal phase of the cycle and maintained in organ culture without hormonal supplementation for up to 5 days. The structure, cell dynamics and steroid-producing capacity of the follicles were compared with those of non-atretic follicles of similar size. The granulosa layer of the atretic follicles invariably regenerated in culture, increasing in thickness more than 2- and 4-fold in large and small follicles respectively. This could not be accounted for by cell division which remained low throughout the culture period. In contrast, non-atretic follicles showed high mitotic activity during the first 24 h in culture: this was not associated with an increase in granulosa thickness in large follicles although there was a 4-fold increase in small ones. An increase in internuclear spacing, a measure of cell size plus intercellular space, partly accounted for the increase in granulosa thickness in atretic follicles. Even when granulosa cells remained in close apposition there was an almost total absence of gap junctions, a prominent feature in the granulosa of non-atretic follicles both in vivo and in vitro. Pyknotic nuclei and atretic bodies rapidly disappeared from the regenerating granulosa layer. The theca interna was restored in culture to a state ultrastructurally closely resembling that of non-atretic follicles in vivo. Total steroid secretion (oestradiol-17beta, testosterone plus progesterone) into the culture medium (pmol.mg tissue-1.24 h-1) was the same for atretic and non-atretic follicles of comparable size. There was, however, a marked difference in the type of steroid produced, largely related to a loss of aromatizing capacity in atretic follicles. The predominant steroid secreted by large non-atretic follicles was oestrogen, with slightly smaller amounts of testosterone, whereas the principal steroid secreted by large atretic follicles was progesterone. In small non-atretic and atretic follicles, the predominant steroid was testosterone, but the non-atretic follicles also secreted appreciable amounts of oestrogen. Addition of FSH to the culture medium did not restore aromatizing capacity to the atretic follicles.     

Alteration of follicular steroid secretion and thecal morphology after in-vitro exposure of individual pig follicles to follicle regulatory protein

Medium-sized (4-6 mm) pig follicles were incubated for 10 h and then examined via light microscopy. Treatment with pig FSH resulted in significantly increased concentrations of oestradiol, testosterone, androstenedione and progesterone in the medium. Follicle regulatory protein (FRP) alone (1 micrograms/ml) decreased follicular secretion of oestradiol (56%) and progesterone (53%) but stimulated the secretion of testosterone (226%) and androstenedione (139%). In the presence of 1 ng FSH/ml, the inhibitory effect of FRP on oestradiol secretion was enhanced (74%), progesterone values were unaffected and secretion of testosterone and androstenedione were reduced by 66% and 53%, respectively. All effects of FRP were fully overcome by 1 micrograms FSH/ml. The incidence of atresia, as defined by granulosa cell pycnosis, was similar in all treatment groups (1-3 of 10 follicles per group). The remaining follicles had intact granulosa cells. However, follicles treated with FRP (1 micrograms/ml) + FSH (1 ng/ml) had pycnotic nuclei in the theca interna cells, in the presence of an intact stratum granulosum. External exposure of follicles to FRP may not reflect physiological conditions since, in vivo, thecal pycnosis is never observed before granulosa cell pycnosis. However, the present results indicate that FRP is potentially capable of altering both follicular morphology and steroidogenesis. We suggest that FSH and FRP interact to affect follicular development.     

Dose-related effects of luteinizing hormone on the pattern of steroidogenesis and cyclic adenosine monophosphate release in superfused preovulatory rat follicles

The secretion of steroids and the release of cAMP in response to repeated luteinizing hormone (LH) stimulation were examined during superfusion of isolated preovulatory rat follicles. A high dose of ovine LH (1 microgram/ml for 20 min) caused a prolonged increase in the secretion of progesterone (P) and 20 alpha-dihydroprogesterone (20 alpha-OHP) and a transient increase in the secretion of testosterone (T) and estradiol-17 beta (E2), and was accompanied by a peak of cAMP release. A single pulse of LH at a low dose level (10 mg/ml for 20 min) gave a limited increase in T secretion, but no clear change in P, 20 alpha-OHP and E2 secretion or cAMP release. When the follicles were challenged with a second pulse of LH (at 1 microgram/ml), the response varied according to the dose of LH delivered in the preceding pulse. Following exposure to the high dose of LH, the follicles were partially refractory to the second LH challenge in terms of cAMP and P and the secretion of T and E2 remained low. The low dose of LH, however, had a conditioning effect on the follicles since the response to the second LH challenge was amplified in terms of P, 20 alpha-OHP and cAMP. In this case a secondary increase in T and E2 secretion was found. The differential response to varying doses of LH are likely to reflect the physiological control of steroidogenesis during final follicular maturation.     

Inhibitory effects of the porcine follicular fluid on estradiol and progesterone secretion by cultured rat granulosa cells

The effect of porcine follicular fluid on estradiol and progesterone secretion was examined using a rat granulosa cell culture with FSH and testosterone in the medium. Follicular fluids from small (less than 5 mm) and large (greater than 6 mm) follicles (SFFI, LFF1) were treated with charcoal, and then fractionated by filtration through an Amicon XM-50 and an PM-10 membrane. The addition of 25% SFF1 and LFF1 into the culture system significantly inhibited estradiol and progesterone secretion (P less than 0.005). These inhibitory activities were observed in PM-10 retentates (10,000-50,000 MW) and filtrates (less than 10,000 MW) of SFF1 and LFF1. The addition of XM-50 filtrates (less than 50,000 MW) of SFF1 and LFF1 caused a dose-dependent inhibition of estradiol and progesterone secretion. The dose-response relationship between the filtrates and estradiol secretion was linear with a significant correlation coefficient. The addition of the filtrates exerted no inhibitory effect on the growth of the cells cultured. XM-50 filtrate of LFF1 from a batch with a low ratio of small/large follicles showed a lower inhibitory activity on estradiol secretion than that of LFF1, while the inhibitory activities in both filtrates on progesterone secretion were almost equivalent. These results suggest that the follicular fluid of small porcine follicle contains nonsteroidal regulators capable of inhibiting estradiol and progesterone secretion by cultured rat granulosa cells, and that the estradiol secretion inhibitor activity decreases in the fluid of large follicle while the progesterone secretion inhibitor activity does not decrease in it.     

Follicle-stimulating hormone regulation of cytochrome P-450 side-chain cleavage messenger ribonucleic acid accumulation by porcine granulosa cells isolated from small and medium follicles

The experiments described here were conducted to examine regulation of cytochrome P-450 side-chain cleavage (SCC) mRNA accumulation in porcine granulosa cells isolated from small (1-4-mm) and medium (5-6-mm) follicles. Granulosa cells were cultured under the following conditions: 1) for 48 h or 96 h with 0, 50, or 200 ng/ml porcine FSH; 2) for 96 h with 200 ng/ml FSH and aminoglutethimide (100 microM); and 3) for 96 h with forskolin (100 microM). Total RNA was extracted and examined by Northern and dot-blot hybridization analysis, and culture media were assayed for progesterone concentration. Northern blot analysis revealed a single band approximately 2.1 kb in size. Accumulation of SCC mRNA by granulosa cells was both FSH dose- and culture time-dependent (p less than 0.05) with maximal increases approximately 4.5 times control levels. Aminoglutethimide reduced progesterone production by about 80% while having no effect on granulosa cell accumulation of SCC mRNA compared to cells stimulated with 200 ng/ml of FSH. Forskolin-treated cells produced significantly more progesterone than did cells treated with FSH, but accumulation of SCC mRNA was similar. In response to FSH, concentration of SCC mRNA did not vary with follicle size, but granulosa cells from small follicles produced significantly more progesterone than did those from medium follicles. These results demonstrate that concentration of SCC mRNA in cultured porcine granulosa cells is FSH dose-dependent, does not vary significantly in cells from small- and medium-sized follicles, and is correlated with progesterone production, but may not parallel progesterone secretion. This last observation indicates that control at sites other than SCC mRNA can affect progesterone production.     

Response of porcine theca and granulosa cells to GH during short-term in vitro culture

In Experiment 1, the influence of exogenous GH on steroid secretion by granulosa and theca interna cells recovered from small (1-3 mm), medium (4-6 mm) and large (8-12 mm) follicles was tested. In the second experiment, theca cells (Tc) and granulosa cells (Gc) obtained from large follicles were cultured separately or in two types, Tc/Gc co-culture, where both types of cells were mixed in one well or Gc and Tc were separated by cell culture membrane inserts. In the third experiment, the influence of GH on the morphology of Gc and Tc cells and activity of Delta(5),3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied. Cells were grown in the control medium (M199+5% of calf serum) or supplemented with 100 ng/ml GH. Testosterone (10(-7) M) was added as the aromatase substrate to granulosa cells cultures. The media were assayed after 48 h of culture for progesterone and oestradiol by RIA. GH added to the culture media had no effect on oestradiol and progesterone secretion by granulosa cells isolated from small and medium follicles while it stimulated both oestradiol and progesterone secretion by Gc isolated from large preovulatory follicles. A stimulatory effect on oestradiol secretion by Tc isolated from all size follicles was observed. GH did not stimulate progesterone secretion by Tc isolated from small follicles but stimulated progesterone secretion by Tc isolated from medium and large preovulatory follicles. Both co-culture systems exhibited synergistic effect on oestradiol secretion. The stimulatory effect on progesterone secretion under the influence of GH was observed in Gc cultured alone and Tc cultured alone. In contrast, the secretion of progesterone was attenuated in both co-culture systems and the addition of GH further augmented this attenuation. A statistically significant increase in oestradiol secretion was observed in all culture conditions. The addition of GH to the culture medium stimulated the activity of 3beta-HSD compared with the control culture from both types of cells. In conclusion, the present studies indicate that there are direct and follicular development stage dependent actions of GH on steroidogenesis of porcine follicular cells.     

In vitro effect of leptin on steroids' secretion by FSH- and LH-treated porcine small, medium and large preovulatory follicles

The role of exogenous leptin in the follicular steroidogenesis in pigs has not been fully elucidated and available data are controversial. In the current study porcine follicles were recovered from ovaries during early, middle, and preovulatory stages of the follicular phase of the estrous cycle. Follicles were cultured in the presence of the recombinant ovine leptin (oLEP) with or without LH (100 ng/ml) or FSH (100 ng/ml). Medium estradiol (E(2)), testosterone (T) and progesterone (P(4)) concentrations were determined after 48h of culture. Leptin at a dose of 2 ng/ml had no effect on basal E(2) and T secretion by small and medium follicles but decreased E(2) secretion by large follicles. Significant synergistic action of FSH and leptin resulting in a 2 - 5 fold stimulation of E(2) secretion by small and medium follicles was observed. The aromatase inhibitor, CGS 16949A augmented T secretion and inhibited E(2) secretion by control and FSH-treated medium follicles. In FSH and leptin-treated follicles, the inhibitory action of CGS 16949A on E(2) secretion was observed. However, there was no augmentation of T secretion. In leptin-treated follicles the stimulatory action on P(4) secretion was observed only during the preovulatory stage. In these follicles, significant synergistic action of leptin with LH on P(4) secretion was also noted. These results indicate that there is a maturation-dependent action of leptin on both E(2) and P(4) secretion. They also suggest a synergistic action of leptin and FSH on E(2) secretion by small and medium follicles as well as leptin and LH on P(4) secretion by large follicles in pigs.     

Changes in the levels of pituitary and steroid hormones in ovine and human ovarian follicular fluid

Changes in the protein and steroid hormones of follicular fluid, aspirated from different follicles of sheep and human ovaries, have been measured and correlated with the size of the follicles. As the fluid contains a number of proteins, steroids have been measured directly and after ether extraction. The follicular fluid concentrations of progesterone and 17 beta-oestradiol measured directly in the fluid increased with the size of the follicles. The levels of free testosterone remained constant in all sizes of follicles, while those of bound hormone showed a 10- to 15-fold increase over the free testosterone concentrations in both the sheep and human follicular fluid. A decrease in the levels of bound testosterone in the fluid of large follicles (LFFL) coincided with the increase in bound 17 beta-oestradiol, suggesting the possible conversion of bound testosterone to oestrogen as the follicle attained maturity. The ratio of follicle-stimulating hormone (FSH) to luteinizing hormone (LH) varied in the fluid obtained from different size follicles, being 1:7 in small (SFFL), 1.3.5 in medium (MFFL) and 1:2.3 in large (LFFL) follicles of sheep ovaries. The LH content of follicular fluid of different size follicles appeared to be the same, with LFFL showing a minor increase over SFFL. In the human, the fluid from medium follicles contained very little LH compared to LFFL. These differences in the pattern of LH levels present in the fluid from different size follicles between human and sheep ovaries presumably reflect species variations in the entry of LH into the follicles.     

Effect of melatonin on steroid production by rat adrenals under in vitro superfusion conditions

In vitro superfusion of adrenals from male and female Wistar rats resulted in a gradual decline of the corticosterone content in the samples collected for a period of 80 minutes. ACTH administration either in the beginning or in the end of the perfusion period led to a marked increase in the level of this steroid. Contrary to this, progesterone content in the collected perfusates was constant throughout the experiments and was not influenced by ACTH. The quantities of testosterone secreted under these conditions were below the sensitivity of the assay. Perfusion with melatonin (200 pg/ml) did not affect corticosterone secretion, but resulted in a significant increase of the progesterone content in the recovered fractions. This effect was immediate and was followed by a decline in the progesterone concentration, observed during melatonin perfusion.     

Decreased luteinizing hormone-stimulated progesterone secretion by preovulatory follicles isolated from cyclic rats treated with the progesterone antagonist RU486.

Since administration of the antiprogesterone RU486 to cyclic female rats at metestrus and diestrus results in increased serum levels of LH, estradiol, and testosterone at proestrus, we investigated whether RU486 affects follicular steroidogenesis. Female rats with a 4-day estrous cycle, induced experimentally by a single injection of bromocriptine on the morning of estrus, were given RU486 (2 mg) twice daily (0900 and 1700 h) on metestrus and diestrus. At proestrus the preovulatory follicles were isolated and incubated for 4 h in the absence and presence of LH. In the absence of LH, accumulation of estradiol, testosterone, and progesterone in the medium was not different for RU486-treated rats and oil-treated controls. In contrast, LH-stimulated estradiol, testosterone, and progesterone secretions were significantly lower in RU486-treated rats compared with controls. Addition of pregnenolone to the incubation medium resulted in a significantly lower increase of progesterone in follicles from RU486-treated rats compared with those from oil-treated controls. This suggests that 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity is decreased by administration of RU486 in vivo. Aromatase and 17 alpha-hydroxylase/C17-20 lyase activities were not affected: addition of substrate (androstenedione and progesterone respectively) did not affect differently the amount of product formed (estradiol and testosterone) in RU486- and oil-treated rats. However, LH-stimulated pregnenolone secretion was lower in follicles from RU486-treated rats compared with follicles from oil-treated controls, suggesting that either cholesterol side-chain cleavage activity or LH responsiveness is decreased. At proestrus the preovulatory follicles from RU486- and oil-treated rats were not morphologically different.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reproductive endocrinology of the Wandering Albatross Diomedea exulans in relation to biennial breeding and deferred sexual maturity

The reproductive endocrinology of the Wandering Albatross Diomedea exulans was studied at South Georgia to investigate the potential endocrine correlates of biennial breeding and of the acquisition of sexual maturity. Gonads of breeding birds and of known-age immature birds of both sexes were examined by laparoscopy throughout the period that they were at the nest site. Blood samples, subsequently analysed to determine concentrations of luteinizing hormone (LH), prolactin, progesterone, testosterone and oestradiol-17/i, were obtained from samples of breeding birds of both sexes at regular intervals from first arrival until the chicks fledged nearly a year later. Before laying in December, breeding birds had mature testes and ovarian follicles and high concentrations of LH, prolactin and sex steroids. Gonadal regression and a rapid drop in hormone levels (except for LH in females) occurred in early incubation (January). Testes (and follicles to a lesser extent) enlarged in mid-incubation, coinciding with high levels of LH and increases in prolactin and testosterone. Gonads finally regressed completely near hatching time. LH, prolactin and testosterone remained at low levels throughout chick rearing (April to November), but females had several periods of active progesterone and oestradiol secretion, and progesterone was detectable in males only late in the chick-rearing period. Although some changes in hormone levels are difficult to explain, the patterns are fairly typical of temperate birds. The persistence of progesterone secretion in both female breeders and non-breeding ‘immature’ birds is viewed as part of a mechanism inhibiting an ovary from becoming vitellogenic. Although testis size and testosterone concentrations increased with age in immature males (of ages 4–10 years), birds of 5 years and older are probably physiologically mature, even though breeding does not start until they are 7 years of age and only half an age group has bred by an age of 11 years. Immature females (of age 4–7 years) had undeveloped follicles, very low oestradiol concentrations but high progesterone levels, providing further support for the role of this hormone in inhibiting gonadotropin secretion. The condition of the female is therefore probably decisive in determining when a pair first attempts to breed but it is unknown what factors initiate normal ovarian development.     

Active immunization of rabbits against progesterone: increase in hormone levels, and changes in metabolic clearance rates and in genital tract tissues

11 alpha-Hemisuccinyl progesterone was coupled to rabbit serum albumin and injected into intact male rabbits, and into intact ovariectomized female rabbits, for a period of 52 weeks. The ovariectomized females and the intact males showed better immune responses than the intact females. Specificity of the antisera was tested against both progesterone and the carrier protein, and against 19 other selected steroids. Antibody titres and serum levels of progesterone in all groups, and testosterones in the males, showed characteristic changes with a first maximum 12-18 weeks after the beginning of immunization, a decrease despite booster injections and a second increase after 35-40 weeks. Hormone levels were 5-12 times higher by the end of immunization than before. High antibody titres were correlated with decreased metabolic clearance rates for progesterone, reflecting the binding of the steroid to the antibody. Increased production rates of progesterone in the immunized females and of testosterone in the immunized males showed that the antibody-bound hormone was not available for feedback control. Absence of primordial follicles, hyperplasia of the Leydig cells, decreased spermatogenesis and involution of the seminal vesicle epithelium were interpreted as direct or indirect effects of the removal of free progesterone.     

Steroid hormone concentrations in the fluid of bovine follicles relative to size, quality and stage of the oestrus cycle

Eight hundred and seven bovine antral follicles from 2 mm to 20 mm in diameter were dissected free of stromal tissue, measured, qualified and divided into 36 groups according to size, quality and stage of cycle. The follicular fluid was collected and assayed by RIA for oestradiol-17beta, testosterone and progesterone. The steroid hormone concentrations vary with follicular size, degree of atresia and stage of the cylce. Non-atretic follicles of less than 8 mm are generally androgen-dominated and non-atretic follicles of more than 11 mm are oestrogen-dominated. Follicles betwen 8 mm and 11 mm are intermediate in this respect. Degeneration leads to a gradual decrease of oestradiol-17beta and testosterone concentration and increase of progesterone. It is suggested that the ratio of oestradiol-17beta/testosterone and oestradiol- 17beta/progesterone and oestradiol-17beta/testosterone + progesterone cannot generally be used to discriminate between non-atretic and atretic follicles. Large follicles present during the early luteal stage contain as much oestradiol-17beta in the follicular fluid as large follicles during the follicular stage, whereas large follicles of the luteal stage contain only 15% of the maximal amount of the latter's. This and other presented data support the statement that follicles present during the early luteal, late luteal and follicular stages of the cycle belong to different groups of growing follicles. It has been concluded that groups of macroscopically qualified follicles can be distinguished from each other by the steroid hormone concentration in the follicular fluid. It is therefore possible to predict the hormonal environment of the oocyte in any individual follicle of a defined size and quality.     

Interleukin-1 alpha increases thecal progesterone production of preovulatory follicles in cyclic hamsters

Adult cyclic hamsters were used to study the effects of interleukin-1 alpha (IL-1 alpha) on in vitro steroidogenesis in preovulatory follicles. IL-1 alpha increased progesterone secretion by preovulatory follicles during a 24-h incubation in RPMI-1640 medium containing hCG (100 mIU/ml) (progesterone levels: 17.5 +/- 2.2 vs. 10.6 +/- 1.9 ng/follicle/ml, p less than 0.05). IL-1 alpha alone had no effect on follicular steroidogenesis. The source of increased progesterone secretion was the thecae (9.8 +/- 1.0 vs. 5.8 +/- 0.4 ng/2 thecae/ml, p less than 0.01) and not the granulosa cells (6.6 +/- 0.2 vs. 6.8 +/- 0.5 ng/20,000 viable granulosa cells/ml). IL-1 alpha also stimulated production of testosterone in thecae of preovulatory follicles. The follicular progesterone increase was dependent on the time of incubation and dose of IL-1 alpha. IL-1 alpha at 5-50 U/ml maximally stimulated progesterone production in the preovulatory follicles, and no significant effect of IL-1 alpha was observed until the 12th hour of incubation. The effects of IL-1 alpha on in vitro steroidogenesis in preantral follicles, experimentally induced atretic preovulatory follicles, and newly formed corpora lutea were examined. IL-1 alpha in the presence of hCG also significantly increased progesterone secretion by atretic preovulatory follicles. In the incubation of preantral follicles or newly formed corpora lutea, however, IL-1 alpha did not alter steroidogenesis. These results indicate that IL-1 alpha stimulates progesterone secretion by preovulatory follicles and that the target tissue for this effect is the thecal layer.(ABSTRACT TRUNCATED AT 250 WORDS)     

The physiological role of beta-endorphin in porcine ovarian follicles

Beta-endorphin-like immunoreactivity (beta-END-LI) was measured by radioimmunoassay in porcine ovarian follicular fluid (FF) from small, medium and large follicles throughout the oestrous cycle. The concentration of beta-END-LI in FF from small follicles collected on days 1-5 of the cycle was at least tenfold higher than in the fluid from any other follicles independently from their size and the period of the cycle. The level of beta-END-LI in small follicles on days 6-10 was drastically decreased. Subsequently, on days 11-16 its concentration was enhanced and reduced again in pre-ovulatory period of the cycle. Concentrations of beta-END-LI in FF from medium follicles were relatively equal throughout the cycle (days 6-21). No significant differences in beta-END-LI levels were found between small, medium and large follicles from days 17-21. However, beta-END-LI concentrations in medium follicles on days 11-13 and 14-16 were statistically lower than those in small follicles. Moreover, the effects of FSH, prolactin (PRL), progesterone (P4), testosterone (T) and 17 beta-oestradiol (E2) on beta-END-LI release by granulosa cells (GCs) from large follicles and, on the other hand, the effects of the opioid agonist FK 33-824 alone or in combination with FSH, PRL or naloxone (NAL) on follicular steroidogenesis were studied. FSH drastically increased beta-END-LI output in a dose-dependent fashion. This stimulatory effect of the gonadotrophin was inhibited by the highest dose of P4 (10(-5) M). The effect of PRL and the steroids added to the cultures on beta-END-LI release was negligible. FSH- or PRL-induced P4 secretion by GCs was essentially abolished by both FK 33-824 and NAL. However, androstenedione (A4) and testosterone output by the cells was greatly potentiated by FK 33-824. In the presence of NAL, FSH or PRL, A4 release stimulated by FK 33-824 was suppressed to the basal level. Secretion of E2 was completely free from the influence of FK 33-824 or NAL; only oestrone (E1) output was modulated by them in cultures where FSH or PRL was present. In conclusion, FSH appears to be the key regulator of beta-END-LI secretion by porcine granulosa cells. Moreover, steroidogenesis in pig granulosa cells is modulated by opioid peptides acting both alone and by way of interaction with FSH or PRL.     

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.     

A comparison of the actions of stimulatory follicular fluid and gonadotropin-releasing hormone analogs on progesterone secretion by porcine granulosa cells

Granulosa cells from small and medium porcine follicles (1-5 mm) were incubated with charcoal-treated follicular fluid from large (6-10 mm) follicles or porcine serum in the presence and absence of gonadotropin-releasing hormone (GnRH) analog and luteinizing hormone (LH) or follicle-stimulating hormone (FSH). A GnRH agonist inhibited follicular fluid's enhancement of basal and LH-stimulated progesterone secretion but did not block follicular fluid's enhancement of FSH-stimulated progesterone secretion. A GnRH antagonist mimicked follicular fluid's enhancement of basal and LH-stimulated progesterone secretion but did not mimic follicular fluid's action on FSH-stimulated progesterone secretion. When the GnRH antagonist and follicular fluid were added together, they acted synergistically in stimulating basal progesterone secretion, and were additive in enhancing LH-stimulated progesterone secretion. These observations suggest that separate follicular fluid molecules are responsible for its influence on LH and FSH actions on granulosa cells and that a GnRH-antagonist-like molecule could be responsible for some of follicular fluid's "luteinization stimulatory" action. Alternatively, the stimulatory follicular fluid molecule may not resemble GnRH but may act via a mechanism that is opposed by GnRH.     

Effect of FSH and HCG on progesterone secretion by cultured oocyte-cumulus complexes and granulosa cells from porcine antral follicles: Influence of follicular development

Oocyte-cumulus complexes and granulosa cells were harvested from small (1–2 mm), medium (3–5 mm), and large (6–12 mm) porcine antral follicles and cultured for 2 and 3 days. The effects of various doses of purified hCG and human FSH on progesterone secretion and monolayer formation were examined. After a 2-day culture period it was found that FSH was more effective in stimulation of progesterone secretion by cultured oocyte-cumulus complexes than in granulosa cells harvested from small follicles (P < 0.01), whereas hCG was more effective in stimulating progesterone secretion in granulosa cells than in oocytecumulus complexes harvested from large follicles. In contrast, after a 3-day culture period, granulosa cells secreted more progesterone compared to oocytecumulus complexes under control conditions or in the presence of hCG or FSH. After 3 days both FSH and hCG stimulated progesterone secretion by oocytecumulus complexes and granulosa cells; however, the hormone effect was greater upon granulosa cells than oocyte-cumulus complexes. After 3 days of culture in the case of both follicular cell types, there was a greater response to FSH in the case of cells harvested from small compared to large follicles. The reverse was true in the case of hCG responsiveness. Monolayer formation ability of oocyte-cumulus complexes was greater in the case of complexes harvested from small and medium than complexes harvested from large follicles. Addition of hCG to the cultures led to a dose-dependent decrease in monolayer formation by oocyte-cumulus complexes harvested from all sizes of follicles.     

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.