Hormonal regulation of progesterone secretion by cultured mouse granulosa cells

Secretion of progesterone by granulosa cells from preovulatory follicles of mice was determined during 2 weeks of cell culture in the presence of androgens, estrogen and pituitary gonadotropins. Androstenedione (10(-7) M) and dihydrotestosterone (10(-7) M) stimulated (P less than 0.05) progesterone secretion during the first 11 days of culture. In contrast, 17 beta-estradiol (10(-11)-10(-7) M) did not alter (P greater than 0.10) progesterone secretion throughout the culture period. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) stimulated (P less than 0.01) the granulosa cells in a dose-dependent manner during the first few days of culture. This luteotropic effect was rapidly lost and at later times when FSH was not effective, LH suppressed (P less than 0.05) progesterone secretion. In the presence of prolactin (Prl) (1 microgram/ml), granulosa cells progressively secreted more progesterone during the first week of culture. After maximal stimulation on Days 7-9, progesterone secretion by Prl-treated cells began to decline, but the amount of steroid produced on Day 13 was still higher (P less than 0.05) than in control cultures. Androstenedione and Prl gave an additive effect on progesterone secretion during Days 3-5 of culture. Thereafter, the androgen, although stimulatory by itself, did not influence the luteotropic action of Prl. Unlike the early effect of androgens, 17 beta-estradiol acted synergistically with Prl to maintain maximal secretion of progesterone during the last 4 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Estrogen stimulation of progesterone synthesis by porcine granulosa cells in culture

Estrogen stimulates synthesis of progesterone by porcine granulosa cells in tissue culture. This enhancement is inhibited by cycloheximide, suggesting that protein synthesis is required for the effect. Estrogen is synergistic with hCG in increasing progesterone synthesis. Sequential treatment with hCG followed by estradiol causes maximal stimulation of progesterone production.     

Arylsulfatase B regulates interaction of chondroitin-4-sulfate and kininogen in renal epithelial cells

The enzyme arylsulfatase B (N-acetylgalactosamine 4-sulfatase; ASB; ARSB), which removes 4-sulfate groups from the nonreducing end of chondroitin-4-sulfate (C4S;CSA) and dermatan sulfate, has cellular effects, beyond those associated with the lysosomal storage disease mucopolysaccharidosis VI. Previously, reduced ASB activity was reported in cystic fibrosis patients and in malignant human mammary epithelial cell lines in tissue culture compared to normal cells. ASB silencing and overexpression were associated with alterations in syndecan-1 and decorin expression in MCF-7 cells and in IL-8 secretion in human bronchial epithelial cells. In this report, we present the role of ASB in the regulation of the kininogen–bradykinin axis owing to its effect on chondroitin-4-sulfation and the interaction of C4S with kininogen. Silencing or overexpression of ASB in normal rat kidney epithelial cells in tissue culture modified the content of total sulfated glycosaminoglycans (sGAGs), C4S, kininogen, and bradykinin in spent media and cell lysates. Treatment of the cultured cells with chondroitinase ABC also increased the secretion of bradykinin into the spent media and reduced the C4S-associated kininogen. When ASB was overexpressed, the cellular kininogen that associated with C4S declined, suggesting a vital role for chondroitin-4-sulfation in regulating the kininogen–C4S interaction. These findings suggest that ASB, owing to its effect on chondroitin-4-sulfation, may impact on the kininogen–bradykinin axis and, thereby, may influence blood pressure.Because ASB activity is influenced by several ions, including chloride and phosphate, ASB activity may provide a link between salt responsiveness and the bradykinin-associated mechanism of blood pressure regulation.     

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.     

Stimulation of progesterone secretion by cultured human granulosa cells with melatonin and catecholamines

Granulosa cells, aspirated from the follicles of patients undergoing treatment for in-vitro fertilization, were cultured in serum-supplemented medium. Adrenaline and noradrenaline stimulated a dose-related increase in progesterone secretion with a maximum stimulation at 10(-5) M, a response that was prevented by the beta-antagonist, propranolol. Adrenaline and hCG showed similar characteristics in their stimulation of progesterone secretion but there was no further increase in progesterone when the 2 compounds were added together. Melatonin stimulated progesterone secretion and, like adrenaline, this stimulation was prevented by propranolol. The ability of both adrenaline and melatonin to increase progesterone secretion was dependent on the degree of follicular development, as determined by peripheral oestradiol concentrations, on the day of laparoscopy. These results suggest that adrenaline and melatonin may have a physiological role in modulating luteal function and that melatonin may act by a beta-adrenergic-related mechanism.     

Enhanced multiplication and progesterone production by porcine granulosa cells cultured in a serum-free medium

Granulosa cells harvested from pro-estrous follicles of porcine ovary were grown in medium 199 supplemented with 0.4, 1 and 10% of growth-promoting calf serum proteins (GPP), and their multiplication and hormonal activity were compared with those of sister cultures carried in medium 199 supplemented with 10% calf serum. The medium containing the growth-promoting proteins was always superior to the whole-serum medium with regard to cell multiplication, activity of Δ5,3 hydroxysteroid dehydrogenase detected histochemically in the cells, and production of progesterone estimated by radioimmunoassay in the medium. It was inferior when it came to estrogen secretion in the beginning of the cultivation when calculated on a per cell basis.     

X-ray analysis of the conformation of chondroitin-4-sulfate calcium salt

The conformation of the chondroitin-4-sulfate calcium salt was investigated by X-ray analysis. The following results were obtained. 1, The repeat length per disaccharide was 0.913 nm: 2, The molecular chain had three-fold screw symmetry: 3, The shape of the unit cell was a trigonal prism with dimensions a=b=1.28 nm, c=2.74 nm, and gamma=120 degrees: 4, The number of disaccharide residues in the unit cell was six. Two molecular chains were packed in the unit cell.     

Hormone-stimulated secretion of luteinization factor in porcine granulosa cells.

Luteinization stimulator (LS) is an intrafollicular compound which was shown to be released by granulosa cells under in vitro conditions with stimulatory effects on immature granulosa cell differentiation. This study was undertaken to determine the effects of various endocrine agents which are involved in the regulation of ovarian function on LS secretion by porcine granulosa cells isolated from 5-8-mm follicles (LGC). Cell conditioned media (CM) obtained after the 4-day culture of LGC were tested in the culture of immature (small) granulosa cells (SGC). The activity of LS released into the LGC conditioned medium was estimated by measuring progesterone (P4) produced by SGC in the presence of CM. Stimulation of P4 secretion was observed after addition of media from cultures treated by LHRH (10(-4) mol.l-1), epinephrine (10(-5) mol.l-1), LH (1 microgram.ml-1), dbcAMP (0.5 and 2.0 micrograms.ml-1) or insulin (1.0-5.0 micrograms.ml-1). Norepinephrine (10(-5) and 10(-7) mol.l-1), estradiol (0.1 and 1.0 microgram.ml-1) and prolactin (0.1 and 1.0 microgram.ml-1) did not change steroidogenic activity of CM. Epinephrine and norepinephrine (10(-5) and 10(-7) mol.l-1), LH (1 microgram.ml-1), dbcAMP (2.0 microgram.ml-1) and estradiol (1 microgram.ml-1) alone enhanced P4 production by SGC, whereas LHRH (10(-3) and 10(-4) mol.l-1), insulin (1.0-5.0 microgram.ml-1) and prolactin (0.1 and 1.0 microgram.ml.-1) did not have any effect. These observations suggest that the process of LS secretion in developing follicles is subject to a specific hormonal control.     

Follicular fluid glycosaminoglycans inhibit degradation of low-density lipoproteins and progesterone production by porcine granulosa cells

Porcine granulosa cells were incubated with commercially available glycosaminoglycans (GAGs) or GAGs purified from porcine follicular fluid to evaluate the effects of GAGs on degradation of low-density lipoproteins (LDL) and progesterone production. Commercially available heparin and chondroitin sulfates (CS) as well as follicular CS and heparan sulfate (HS) inhibited degradation of LDL in dose-dependent manners. Doses of follicular CS and HS required to inhibit 50% of the LDL degradation corresponded to concentrations found in follicular fluid (less than 1 mg/ml). Progesterone production was also inhibited in a dose-dependent fashion by follicular GAGs at concentrations found in follicles. The ability of the follicular GAGs to inhibit degradation of LDL could represent a mechanism by which the utilization of LDL-derived sterol is temporarily restricted following permeabilization of the ovulatory follicle. Follicular GAGs may also modulate utilization of apoprotein E-containing high-density lipoproteins in unruptured follicles.     

Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin

We constructed a human soluble thrombomodulin (sTM) expression vector using the RSV promoter. Recombinant sTM (rsTM) was expressed in CHO cells and was recovered from culture medium by ion exchange chromatography. Two active fractions, designated as rsTM alpha (low salt elution) and rsTM beta (high salt elution), were detected and further purified by immunoaffinity chromatography. Purified rsTM beta contained bound chondroitin-4-sulfate as judged by HPLC detection of the chondroitinase ABC and AC I digestion product, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose. The apparent Kd values for thrombin of alpha and beta were 7.4 and 1.4 nM respectively. RsTM beta was more effective at inhibition of thrombin clotting activity and had antithrombin III-dependent anticoagulant activity which was not possessed by rsTM alpha. Both anticoagulant activities were lost after chondroitinase treatment of rsTM beta.     

Effect of androgens and gonadotropins on progesterone secretion of chicken granulosa cells

A culture system has been used to study the effect of PMSG in vivo pretreatment and androgens on the in vitro secretion of progesterone from avian granulosa cells. PMSG in vivo pretreated cells secreted greater amounts of progesterone than did cells obtained from untreated hens. Testosterone and 5-alpha-dihydrotestosterone significantly increased basal progesterone secretion in PMSG pretreated cells as well as in granulosa cells harvested from non-treated hens. Testosterone or 5 alpha-dihydrotestosterone in combination with FSH or LH were additive and never resulted in a synergistic stimulation of progesterone secretion.     

Melatonin directly stimulates the secretion of progesterone by human and bovine granulosa cells in vitro

Melatonin, at concentrations and periods of exposure reflecting those present during the circadian cycle, was investigated for its influence on steroid production by granulosa cells cultured in serum-supplemented medium. At high (200 pg/ml) but not low (20 pg/ml) physiological concentrations, melatonin significantly stimulated progesterone production by human granulosa cells. This response was independent of the overall level of cell activity and was seen under the different culture conditions associated with different culture media. Exposure to melatonin for 8 h significantly stimulated progesterone secretion to a level similar to that achieved under continuous exposure, and the effect was reduced to control levels during subsequent periods in which no melatonin was added. Melatonin had no consistent effect on aromatase activity in the conversion of stored or serum-available androgen to oestradiol. Melatonin significantly stimulated progesterone production by bovine granulosa cells in vitro, at concentrations similar to those present during the endogenous nocturnal rise (100-400 pg/ml). This response to physiological conditions by human and bovine cells suggests a role for melatonin in the regulation of progesterone production by the ovary.     

Effects of blood monocytes and lymphocytes on progesterone secretion by granulosa cells in the pig

We studied the effects of autologous and nonautologous co-cultures of porcine blood monocytes and lymphocytes with granulosa cells on progesterone secretion. Eight prepubertal crossbred gilts were ovariectomized, and the granulosa cells were collected, plated at 2.5 x 10(5) cells/ml and allowed to attach. Blood was obtained from the same eight gilts, and the mononuclear cells were separated by density gradient centrifugation. Monocytes were separated from lymphocytes by adherence to plastic. Adherent monocytes, lymphocytes and a 1:1 mixture of monocytes + lymphocytes were added to granulosa cell cultures and incubated for 48 h. Progesterone secretion into the media was measured. In addition, blood cell alloreactivity was studied in these co-cultures by measuring uptake of (3)H-thymidine. The co-culture of adherent monocytes or monocytes + lymphocytes with granulosa cells increased (P <.05) progesterone secretion as compared with granulosa cells cultured alone. However, co-culture of lymphocytes with granulosa cells did not have a significant effect. No difference was observed between autologous and nonautologous cell cultures in blood cell proliferation or granulosa cell progesterone secretion. In conclusion, blood monocytes influence progesterone secretion by granulosa cells. In addition, there was no difference in the ability of autologous and nonautologous blood cells to stimulate progesterone secretion by granulosa cells. No alloreactivity was observed using nonautologous immune cells with granulosa cells.     

The influence of prolactin (PRL) on progesterone secretion by porcine luteal cells in vitro

Porcine luteal cells were obtained from corpora lutea on the 5th, 13th and 17th days of the estrous cycle. The cells were suspended at a concentration of 5 × 10⁴ cells/ml in Eagle's medium with 2% human serum albumin. These cells were incubated with or without 0.01, 0.1, 1 or 10 μg/ml porcine prolactin. The amount of progesterone in cultures was estimated by a radio-immunological method after 30 min, 3 h and 6 h of culturing.Luteal cells obtained on the 5th day of the estrous cycle and incubated without prolactin secreted 71.24 ± 21.91 ng progesterone/ml of medium, whereas under the influence of prolactin at 0.01, 0.1, 1 and 10 μg/ml, 39.06 ± 13.33, 44.31 ± 12.69, 44.88 ± 16.85 and 51.62 ± 15.01 ng progesterone/ml (P<0.01) were secreted. Luteal cells from the 13th day of the estrous cycle incubated without prolactin secreted on average 70.72 ± 9.21 ng progesterone/ml of medium, whereas under the influence of different prolactin doses 50.75 ± 8.52, 46.54 ± 7.13, 43.30 ± 6.78 and 41.68 ± 7.21 ng progesterone/ml (P<0.01) were secreted.Prolactin did not change progesterone secretion by luteal cells obtained on the 17th day of the estrous cycle. An influence of the incubation time on progesterone secretion by these cells was observed: after 30 min of incubation the cells secreted 8.83 ± 2.95 ng/ml, after 3 h 8.12 ± 2.57 ng/ml and after 6 h 6.86 ± 1.91 ng/ml, irrespective of the amount of PRL added.The results suggest that prolactin plays a role in the luteolysis of the corpus luteum.     

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.     

Modifications of low-density lipoprotein induced by arterial proteoglycans and chondroitin-6-sulfate

Association of low-density lipoproteins (LDL) with arterial chondroitin sulfate proteoglycans (CSPG) appears to contribute to their deposition in the extracellular intimal compartment and to its internalization by macrophages. CSPG and LDL interact by ionic bridges with formation of soluble and insoluble complexes. We studied the alterations on LDL structure induced by its association with arterial CSPG and other glycosaminoglycans (GAG). In soluble complexes, at low and physiological ionic strength, arterial CSPG and sulfated GAG modify the kinetics of apoB-100 proteolysis by trypsin. However, less marked alterations in the peptide patterns were observed with proteinase V8 and almost none with thermolysin. This is indirect evidence that the presence of CSPG and GAG modified the exposure of polar regions of apoB-100 in LDL. Competitive binding experiments with agarose-bound heparin and soluble GAG also suggest that after formation of insoluble complexes with arterial CSPG and resolubilization the exposure of Lys, Arg-rich segments of apoB-100 is increased. Results from differential scanning calorimetry and differential thermal spectrophotometry showed that the CSPG and GAG-induced modifications reduced the thermal stability of the surface and core in LDL. If present in vivo, the structural alterations of polar segments of the LDL protein moiety may influence the outcome of its alteration with the arterial mesenchyma.     

Secretion of follicle-regulatory protein by porcine granulosa cells

Follicle-regulatory protein (FRP) affects ovarian steroidogenesis and thus follicular maturation. However, secretion of FRP by cells from different-sized follicles as well as the modulation of FRP production by gonadotropins and locally produced steroids are unknown. To evaluate which cell type secretes FRP, theca and granulosa cells were obtained from porcine follicles. In addition, the effects of follicle-stimulating hormone (FSH) and steroids on FRP secretion from granulosa cells of small (less than 3 mm), medium (3-6 mm), and large (greater than 8 mm) porcine follicles and theca cells of large follicles were determined. Granulosa cells were obtained from follicular aspirates, whereas theca cells were recovered after digestion of the stereomicroscopically removed thecal layer. Both were cultured in monolayer in serum-free medium. Granulosa cells were treated as follows: 1) control; 2) FSH (250 ng/ml); 3) progesterone (500 ng/ml, 3 micrograms/ml), or estradiol-17 beta (500 ng/ml, 4 micrograms/ml), or dihydrotestosterone (500 ng/ml, 1 microgram/ml); 4) FSH + progesterone, or estradiol-17 beta, or dihydrotestosterone. Theca cells received the same treatment except that human chorionic gonadotropin (hCG) (5m IU/ml) was used in place of FSH. At 48 or 96 h, media were removed and FRP was quantitated by an Enzyme-Linked Immunosorbent Assay (ELISA). FRP was identified in granulosal medium from follicles of all sizes, but was not present in thecal cultures. At 48 h, granulosa cells from small and medium-sized follicles produced more FRP (20.04 +/- 4.4, 35.42 +/- 4.1 immunoreactive units [IRU]) than cells from large (3.53 +/- 0.97 IRU) follicles.(ABSTRACT TRUNCATED AT 250 WORDS)