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At least 19 recordsLinked to original sources

PubMed1978

[Action of HCG on isolated and cultivated sow ovarian granulosa cells].

Granulosa cells harvested from cavitary follicles of varying diameters have been cultivated in vitro in basic conditions and in presence of HCG, and then tested for the activity of steroid-3 beta-ol-dehydrogenase and observed with a scanning electron microscope. As concerns the enzymatic activity, the cells of the smaller follicles (5--6 mm) show a very low reactivity which increases in presence of HCG. In the cells of the larger antral follicles (8--9 mm) the reactivity is remarkably higher, particularly when HCG is added to the medium. With the scanning electron microscope the appearance of the granulosa cells of the larger follicles cultivated with HCG is very peculiar because of the presence on the cell surface of many cytoplasmic evaginations. These are more numerous and thinner than those normally found on the uncultured cells and on the cells cultivated in basic conditions.

3-Hydroxysteroid Dehydrogenases↗

PubMed1976

Androgens augment FSH-induced progesterone secretion by cultured rat granulosa cells.

Granulosa cells have been isolated from ovaries of estrogen-treated immature intact and hypophysectomized rats, and have been maintained in culture in a chemically-defined medium. Progesterone secretion by these cells was testosterone or 17beta-OH-5alpha-androstan-3-one (DHT), progesterone secretion was low or undetectable. However, the addition of testosterone or DHT together with FSH caused a dramatic 8- to 19-fold increase over that caused by FSH alone. On the other hand, luteinizing hormone (LH) alone had no effect on progesterone secretion, but produced a small stimulation when added together with testosterone. These results demonstrate synergism between androgens and FSH in the control of progesterone secretion by granulosa cells in culture.

Animals↗

PubMedDate not supplied

A synergistic effect of androgen on the stimulation of progesterone secretion by FSH in cultured rat granulosa cells.

Granulosa cells from preovulatory follicles (PO) or from the enlarged preantral follicles of hypophysectomized immature diethylstilbestrol-treated (Hx-DES) rats were cultured with various combinations of FSH, androst-4-ene-3,17-dione (Ad), estradiol-17beta and dibutyryl cyclic AMP (dbcAMP). Progestin levels (progesterone and 20alpha-dihydroprogesterone) in the medium after 2 days of culture were assayed by radioimmunoassay. The control levels of the two progestins were lower for Hx-DES than for PO cells. Rat FSH (NIAMD-1-3;0.1 mug/ml) caused a 2-fold rise in progestin accumulation in both PO and Hx-DES cultures, dbcAMP (1 mM) increased progestin accumulation in PO cultures 4-5-fold, and to an even greater extent (10-20 fold) in Hx-DES cultures. Androstenedione (1.0 mug/ml) augmented progestin accumulation (1.5-3-fold), and synergized the steroidogenic action of FSH: in cells from Hx-DES rats, combined treatment with FSH and Ad caused a 5-10-fold increase over the values obtained with FSH alone. Testosterone and 5alpha-dihydrotestosterone, but not estradiol-17beta or estrone, mimicked these effects of Ad, Ad did not synergize the action of dbcAMP on progestin levels in Hx-DES cultures. It is proposed that androgen may play a role in the development of the FSH-responsive mechanism in preantral granulosa cells.

Androgens↗

PubMed1975

Morphological variations in gap junctions of ovarian granulosa cells.

Granulosa cells in ovarian follicles of rat, mouse, rabbit and hamster were studied by lanthanum tracer and freeze-fracture techniques. Abundant gap junctions exhibited striking intraspecific variation in size and pattern of particle aggregation. The smaller gap junctions showed close packing of the intramembranous A face particles. In large gap junctions, ranging up to 6 mu in diameter, particles were packed in rectilinear arrays separated by a labyrinthine network of particle-free 'aisles'. Small clusters of particles in a particle-poor circumferential zone suggested enlargement of junctions by peripheral accretion. Linear intramembranous structures, resembling those of occluding junctions, occasionally bounded large gap junctions. Spherical intracytoplasmic structures limited by gap junctional membranes were shown by tracer studies to arise by invagination of the cell surface. These were intrepreted as a means of disposal of junctions by interiorization.

Animals↗

PubMed1978

Synthesis of lipoprotein lipase in cultured avian granulosa cells.

Avian granulosa cells cultured as a homogeneous parenchymal population contain lipolytic activity. This activity is stimulated 2--5-fold by serum, inhibited 90% by 1 M NaCl and inhibited 80% by specific anti-lipoprotein lipase immunoglobulins. 85% of the activity binds to heparin-Sepharose 4B, and 70% of bound activity is eluted with 1.5 M NaCl. Thus, the lipolytic activity of cultured granulosa cells is lipoprotein lipase. Granulosa cells were shown to synthesize lipoprotein lipase in culture by incorporating [3H]leucine into the enzyme protein, as measured with an immunoadsorption technique. Finally, colchicine was shown to increase intracellular lipolytic activity, suggesting an inhibition of secretion of this enzyme by cultured granulosa cells.

Animals↗

PubMed1979

A comparison between oocyte growth in coculture with granulosa cells and oocytes with granulosa cell-oocyte junctional contact maintained in vitro.

Evidence is presented that strongly supports the hypothesis that the junctional association between oocytes and granulosa cells must be maintained to promote oocyte growth and development in vitro and that the coculture of oocytes with granulosa cells is not a sufficient condition for oocyte development. Furthermore, it is shown that incorporation of uridine and leucine by oocytes into TCA-insoluble material is significantly greater in granulosa cell-enclosed oocytes than in cocultured oocytes.

Animals↗

PubMed1975

Demonstration of a specific receptor for prolactin in porcine granulosa cells.

Porcine granulosa cells and subcellular fractions from these cells have been shown to have a specific receptor for ovine prolactin (OPRL). Ovine growth hormone demonstrated 7% of the potency of OPRL in displacing 125I-OPRL from its binding site; FSH, TSH, LH, insulin and ACTH showed negligible cross-reactivity. Scatchard analysis of the displacement curves suggested that 125I-OPRL has a high affinity for its receptor with a dissociation constant (Kd) for the granulosa cell-receptor of 7.4-7.7 times 10(-10) M with no change as the follicle enlarges. In contrast, the specific binding of prolactin decreased markedly with maturation of the follicles with an apparent decrease in binding sites/cell from 555 in small follicles to 300 in large (preovulatory) follicles. The demonstrated Kd's were within the range of prolactin concentrations easily attained in vivo and were in good agreement with values obtained in our laboratory and elsewhere for the prolactin receptor from mammary gland and other tissues. Consequently, these studies may provide a basis for a better understanding of the role of prolactin in ovarian function.

Animals↗

PubMed1978

Steroid production from 17alpha-hydroxypregnenolone and dehydroepiandrosterone by human granulosa cells in vitro.

Granulosa cells were aspirated 3--4 h before the expected time of ovulation from 10 follicles of 4 patients treated with gonadotrophins: 4 of the follicles were immediately preovulatory. The granulosa cells were cultured for 10 h with 17alpha-hydroxypregnenolone or dehydroepiandrosterone and samples of medium removed at 3 and 10 h were assayed for 6 steroids. Granulosa cells were unable to synthesize androgens from endogenous substrate or undertake conversions via the delta5 pathway, but cells from all follicles were capable of aromatizing exogenous androgens to oestrogens although this capability was reduced in cells from follicles beginning to luteinize. Granulosa cells from preovulatory follicles synthesized more progesterone from endogenous substrate than cells from follicles which had not begun to luteinize. The results provide further support for the two-cell theory of oestrogen biosynthesis whereby granulosa cells aromatize androgens which are synthesized by the thecal cells in vivo.

17-alpha-Hydroxypregnenolone↗

PubMed1976

Hormonal function of a granulosa cell tumor.

Granulosa and theca cell tumors are rather common gonadal stromal tumors. A postmenopausal patient with a granulosa cell tumor, who complained chiefly of breast tenderness and enlarging, abdomen, is presented. Preoperative and postoperative studies including serum estrone, estradiol, prolactin, FSH, and LH,as well as urinary estrogens, 17-ketosteroids, and 17-hydroxysteroids are reported. A plan of treatment and followup is suggested. It is recommended that survival data on patients with such slow-growing tumors be adjusted to reflect the true incidence of death from the specific tumor in question.

Aged↗

PubMed1978

Maturational changes in sheep ovarian follicles: gonadotrophic stimulation of cyclic AMP production by isolated theca and granulosa cells.

Theca and granulosa tissues isolated from sheep ovarian follicles of different sizes were incubated in the presence of human chorionic gonadotrophin (HCG; 5 IU/ml) or follicle stimulating hormone (FSH; 5 microgram NIH-FSH-S11/ml) for 40 min. Changes in the total amounts of cyclic 3',5'-adenosine monophosphate (cAMP) were used as an index of the responsiveness of these preparations to the hormones. Thecal tissue of both large (4-6 mm in diameter) and small (1-3 mm) follicles responded similarly to gonadotrophins. Granulosa cells from small follicles failed to respond to stimulation by HCG. FSH, however, consistently increased cAMP production in comparison with controls or cells treated with HCG. Granulosa cells of large follicles responded to both HCG and FSH.

Animals↗

PubMed1992

Porcine granulosa cells do not express transforming growth factor-beta 2 (TGF-beta 2) messenger ribonucleic acid: molecular basis for their inability to produce TGF-beta activity comparable to that of rat granulosa cells.

In contrast to rat granulosa cells (GC), GC of the pig and cow produce very low levels of transforming growth factor-beta (TGF-beta)-like activity in vitro. Because cultured rat GC predominantly express TGF-beta 2 messenger RNA (mRNA) and secrete high levels of the protein, we hypothesized that TGF-beta 2 mRNA expression by porcine GC might be absent or diminished, thus providing a molecular explanation(s) for their relatively low levels of TGF-beta production. We tested this hypothesis by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay analysis. When analyzed by RT-PCR, porcine GC RNA from 1-3 mm follicles did not yield the expected 489 base pair (bp) TGF-beta 2 product but instead generated a smaller than anticipated 240 bp species; porcine testis RNA generated both the 240 and the anticipated 489 bp products. Sequencing these species indicated that the smaller form was not a novel TGF-beta 2 splice variant, and that the 489-bp product was porcine TGF-beta 2. This is the first reported nucleotide sequence for porcine TGF-beta 2; it is 90% and 91% identical to murine and human TGF-beta 2 sequences, respectively. Further RT-PCR analysis of porcine GC RNA resulted in the identification of bp products representing TGF-beta 1 and TGF-beta 3 mRNA. Enzyme-linked immunosorbent assay analysis of porcine GC conditioned medium confirmed the presence of TGF-beta 1 at very low levels. TGF-beta 2 was undetectable. Comparable analysis of GC from the diethylstilbestrol-treated prepubertal rat demonstrated the presence of TGF-beta 1 and TGF-beta 3 mRNA by RT-PCR and very low levels of the corresponding protein products in conditioned culture medium. Collectively, these results suggest that the inability of porcine GC to express TGF-beta 2 mRNA could explain the very low levels of TGF-beta activity secreted by these cells in vitro.

Animals↗

PubMed1979

Hormonally induced cell shape changes in cultured rat ovarian granulosa cells.

Cultured rat ovarian granulosa cells undergo a dramatic morphological change when exposed to follicle-stimulating hormone (FSH). Exposure to FSH causes the flattened epithelioid granulosa cells to assume a nearly spherical shape while retaining cytoplasmic processes which contact the substrate as well as adjacent cells. This effect of FSH is preceded by a dose-dependent increase in intracellular cAMP, is potentiated by cyclic nucleotide phosphodiesterase inhibitors, and is mimicked by dibutyryl cAMP. Prostaglandins E1 or E2 and cholera enterotoxin also cause the cells to change shape. A subpopulation of the cells responds to luteinizing hormone. These morphological changes, which are blocked by 2,4-dinitrophenol, resemble those produced by treating cultures with cytochalasin B. Electron microscopy shows that the unstimulated, flattened cells contain bundles of microfilaments particularly in the cortical and basal regions. After FSH stimulation, microfilament bundles are not found in the rounded granulosa cell bodies but they are present in the thin cytoplasmic processes. These data suggest that the morphological change results from a cAMP-mediated, energy-dependent mechanism that may involve the alteration of microfilaments in these cells.

Animals↗

PubMed1976

Androgenic function of a granulosa cell tumor.

Virilizing granulosa cell tumors are uncommon and have not been well studied hormonally. A hirsute woman with a cystic granulosa cell tumor of the ovary is presented. Plasma hormone levles obtained before and after surgery indicate testosterone production by the tumor with LH and FSH suppression. Plasma testosterone (T) and T-index returned to normal after tumor removal, and ovulation resumed.

Adult↗

PubMed1979

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.

Animals↗

PubMed1976

Enzyme histochemistry of cultured ovarian cells. I. Histochemistry of porcine preovulatory granulosa cells in culture.

Histochemical activity of hydroxysteroid dehydrogenases as well as alkaline and acid phosphatase was investigated in porcine granulosa cells cultured in vitro. Granulosa cells, isolated from preovulatory procine ovarian follicles, during in vitro culture showed activity of enzymes participating in steroid biosynthesis. High activity of delta53betta0H-SDH and G6P-DH as well as the activity of alkaline phosphatase, appearing in the course of culture, could be evidence of progressive luteinization of the cells. Activity of 17beta0H-SDH was lower and exhibited strong fluctuations, similarly low was 20xOH-SDH. Gonadotropic hormones caused the increase of synthesis and accumulation of intracellular lipids. They stimulated alkaline and acid phosphatase, and also the activity of the dehydrogenases. LH had the most visible effect. Estradiol stimulated the activity of acid but not alkaline phosphatase and was not influencing, even lowering the activity of dehydrogenases.

Acid Phosphatase↗

PubMed1975

The cytotoxic effect of serum from patients with Addison's disease and autoimmune ovarian failure on human granulosa cells in culture.

Human granulosa cells growing in culture were used to study the effects of sera from twenty-three patients with idiopathic Addison's disease. There were two women with primary amenorrhoea, thirteen with premature ovarian failure following a normal menarche, and one man with testicular atrophy in this group. The sera from all twenty-three patients contained antibodies which reacted with fresh sections of human corpus luteum using an in vitro immunoflorescence test. Serum fron nine of the twenty-three patients contained antibodies which were cytotoxic to human granulosa cells in tissue culture; the cytotoxic effect was complement dependent, and related to the immunofluorescent staining properties of the serum and dilution of antibody present. No cytotoxic effect was observed when granulosa cells were cultured in serum from the remaining fourteen Addisonian patients, or from normally menstruating women, post-menopausal women or in serum containing organ-specific antibodies to other endocrine tissues. The cytotoxic effect was paralleled by a fall in progesterone production by the granulosa cells. The production of progesterone by cells cultured in any of the non-cytotoxic sera was significantly greater than that produced by cells in the presence of cytotoxic antibody.

Addison Disease↗

PubMed1977

Effects of fibroblast and epidermal growth factors on ovarian cell proliferation in vitro. I. Characterization of the response of granulosa cells to FGF and EGF.

Despite numerous studies on the effects of gonadotropins on ovarian cells in tissue culture, the factors controlling the proliferation of granulosa cells in vitro remain unknown. We have examined the effect of fibroblast growth factor (FGF) and epidermal growth factor (EGF) on granulosa cell proliferation in vitro in an attempt to clarify their possible roles in the control of ovarian development. FGF and EGF both stimulate DNA synthesis in resting populations of granulosa cells. The half-maximal response forthis effect with FGF was observed at 4 X 10(-11)M and with EGF at 1.5 X 10(-13)M. Autoradiography demonstrated that the whole cell population initiated DNA synthesis in the presence of either EGF or FGF, thus precluding an additive effect of the two mitogens. When cells were maintained at low density (100 cells/cm2) in the presence of low serum (1%) they divided with a doubling time of 48-72 h, but addition of either EGF or FGF accelerated their proliferation. The doubling time observed in the presence of FGF was 16 h versus 20 h with EGF and the final cell density reached in the presence of EGF or FGF was 20 times that of cells maintained in the presence of 1% calf serum alone. In the presence of 10% serum, granulosa cells had a doubling time of 24 h and the final density reached was similar to that observed in 1% serum with EGF and FGF. Addition of EGF or FGF to 10% serum resulted in a final density 3 to 4-fold higher than that observed with 10% serum alone. The ultrastructure of the granulosa cells grown in the presence of EGF or FGF was similar to that of cells maintained in the absence of added mitogens. The only marked difference was that cells grown in the presence of FGF or EGF had a high lipid granule content while cells grown in their absence had a low lipid granule content. The effect of various concentrations of FGF and EGF on the proliferation of granulosa cells has been analyzed. The minimal effective dose of EGF was 3 X 10(-14)M and saturation was observed at 3 X 10(-11)M, with a half-maximal response at 6 X 10(-13)M. With FGF the minimal dose stimulating proliferation was 1.5 X 10(-12)M and saturation was achieved at 1.5 X 10(-10)M, with a half-maximal response at 3 X 10(-11)M. Our results show that EGF and FGF are the most potent mitogens ever observed and are mitogenic for granulosa cells at 300 to 3000-fold lower concentrations than for other cell types which have been studied, such as fibroblasts or lens epithelial cells.

Blood↗

PubMed1979

Effects of follicular development on the ability of cultured porcine granulosa cells to convert androgens to estrogens.

Porcine granulosa cells from different stages of follicular development were examined for their ability to convert androgens to estrogens and their ability to secrete progesterone. Granulosa cells from all stages of follicular development can convert androgens to estrogens, and the addition of exogenous androgen is required for estrogen secretion. Granulosa cells obtained from medium and large follicles have a greater capacity to convert androgens to estrogens than do granulosa cells obtained from small follicles. The addition of FSH brought about an increase in estrogen secretion in the presence of androgen only in granulosa cells from large follicles. LH attenuated estrogen secretion in granulosa cells from medium follicles treated with testosterone. Granulosa cells from medium follicles were unable to secrete estrogen from days two to four irrespective of treatment. Androgens augment FSH stimulated progesterone secretion in granulosa cells from medium follicles from days two to four, and the addition of follicular fluid from small follicles stimulates progesterone secretion in the presence of FSH and androgens.

Androgens↗