Gonadotrophin-binding components in porcine follicular fluid

1990 ◽  
Vol 124 (3) ◽  
pp. 485-494
Author(s):  
T. A. Yarney ◽  
M. R. Sairam ◽  
G. N. Bhargavi ◽  
B. R. Downey ◽  
A. Srikandakumar
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Binding Sites ◽  
Cell Receptor ◽  
Binding Activity ◽  
Human Chorionic Gonadotrophin ◽  
Supernatant Fraction ◽  
Receptor Binding Activity ◽  
Membrane Bound ◽  
Hcg Receptor

ABSTRACT The follicular fluid is an important milieu for the growing and maturing oocyte and granulosa cells. In this study we investigated: (1) the properties of gonadotrophin-binding sites in the supernatant fraction of porcine follicular fluid (pFF) and compared them with those of membrane-bound receptors, and (2) the relative changes that occur in pFF and granulosa cell receptor-binding activity following hormone priming of gilts. 125I-Labelled human chorionic gonadotrophin (hCG) and 125I-labelled ovine FSH (oFSH) binding to particulate and supernatant fractions of pFF were hormone-specific and saturable. The concentration of 125I-labelled hCG-binding sites was roughly 50-fold higher in particulate than in supernatant fractions of pFF. However, 30–40% of the total 125I-labelled hCG-binding activity in pFF was present in the supernatant fraction of commercial batches of pFF. 125I-Labelled oFSH binding to pFF membranes was markedly higher than to supernatant fractions. Binding of 125I-labelled hCG and 125I-labelled oFSH to granulosa cells and supernatants of pFF showed a time-dependent variation in response to hormone priming. The results suggest that gonadotrophin-binding sites in the supernatant fraction of pFF have properties similar to those of their membrane-bound counterparts. 125I-Labelled hCG-binding activity in the supernatant fraction of pFF was shown to be more stable than detergent-solubilized LH/hCG receptors, even in glycerol-preserved preparations. Based on a number of criteria, we have speculated that pFF may have components which may be similar in structure to the extracellular domain of the LH/hCG receptor. Journal of Endocrinology (1990) 124, 485–494

THE LH-hCG RECEPTOR OF HUMAN OVARY AT VARIOUS STAGES OF THE MENSTRUAL CYCLE

1975 ◽  
Vol 79 (3) ◽  
pp. 568-576 ◽  
Author(s):  
S. Wardlaw ◽  
N. H. Lauersen ◽  
B. B. Saxena
Keyword(s):  
Menstrual Cycle ◽  
Binding Sites ◽  
Receptor Site ◽  
Binding Activity ◽  
Corpora Lutea ◽  
Human Ovary ◽  
Slow Decline ◽  
Number Of Binding Sites ◽  
Human Ovaries ◽  
Hcg Receptor

ABSTRACT Receptors specific for hCG were found in human corpora lutea and follicles. hCG and LH were found to bind at a similar receptor site. The dissociation constant for hCG ranged from 10−10 to 10−11 mol/l in human corpora lutea. The number of binding sites for 125I-hCG ranged from 10−14 to 10−15 moles/mg protein in human corpora lutea. The binding of 125I-hCG to ovary was found to vary at different stages of the menstrual cycle. The binding of 125I-hCG to human ovaries increased on days 13–15 of the cycle, then declined slightly, and increased again on days 22–23. Following day 23, there was a slow decline until day 27 when binding activity could no longer be measured. No binding could be measured by the corpus luteum after the onset of menstruation or in corpora albicans.

Distribution of the β-core human chorionic gonadotrophin fragment in human body fluids

1992 ◽  
Vol 135 (1) ◽  
pp. 175-188 ◽  
Author(s):  
S. F. de Medeiros ◽  
F. Amato ◽  
D. Bacich ◽  
L. Wang ◽  
C. D. Matthews ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Intramuscular Injection ◽  
Body Fluids ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Pregnancy Urine ◽  
In Pregnancy

ABSTRACT The origins of a fragment of the human chorionic gonadotrophin (hCG) molecule, β-core (βC-hCG) were studied by analysis of βC-hCG concentrations in biological fluids. In addition, the ability of the placenta to produce the fragment and the metabolism of hCG to βC-hCG by human granulosa cells was determined in tissue culture. Finally the conversion of exogenous hCG to βC-hCG was studied in vivo. The fragment was present in pregnancy urine as well as that from premenopausal and postmenopausal subjects. The highest concentrations were found in pregnant women. Ratios of βC-hCG to intact hCG were higher in pregnancy urine when radioimmunoassay (RIA) was used compared with immunoradiometric assay (IRMA) (0·67 and 0·37 respectively). Concentrations of βC-hCG were higher in postmenopausal urine than in premenopausal specimens. A significant amount of a high molecular weight βC-hCG immunoreactive material was found in serum samples after size separation, and the molar ratio of βC-hCG/hCG was estimated as 0·019. Amniotic fluid also contained small quantities of two forms of immunoreactive βC-hCG and the ratio of 0·01 for authentic βC-hCG/hCG increased to 0·026 when the high molecular weight form was considered. Cultured trophoblastic tissue released material with βC-hCG immunoreactivity in the medium and chromatographic separation revealed that the majority of this material was of higher molecular weight compared with the authentic βC-hCG form. βC-hCG was the principal glycoprotein found in follicular fluid after hyperstimulated folliculogenesis and intramuscular injection of 5000 IU hCG. We also demonstrated that 26% of follicular fluid samples (n = 50) were positive for βC-hCG; levels ranged from 5·2 to 23·0 pmol/l (13·1 ±5·7); s.d.) when a specific IRMA was used. The RIA could detect βC-hCG in 48 samples (96%), levels ranging from 7·0 to 28·5 pmol/l (19·4±5·2). Moreover, granulosa cells cultured in the presence of hCG were able to degrade the intact molecule to both high molecular weight and authentic immunoreactive forms of βC-hCG. After gel filtration, material of molecular weight over a wide range and immunoreactive for βC-hCG was present in human seminal plasma. Assaying 74 samples of this fluid by IRMA, βC-hCG was detected in 42 (56·7%), levels ranging between 5·5 and 59·5 pmol/l (24·9± 15·2). Following intramuscular injection of 1500 IU hCG into male volunteers, the levels of βC-hCG in urine increased by approximately 220% during the first 24 h (P= 0·036 for βC-hCG levels at 2 h and 24 h), decreasing thereafter to undetectable levels in the next 72 h. However, in serum, βC-hCG immunoactivity remained under the limit of detection of the assay at all times. We concluded that (1) the βC-hCG fragment is widely distributed in body fluids and a dissociable high molecular weight material immunoreactive for βC-hCG is found in some biological compartments; (2) granulosa-lutein cells are able to degrade intact hCG to a small βC-hCG immunoreactive fragment; (3) trophoblastic cells synthesize and release different size material with βC-hCG immunoreactivity; (4) intramuscular injection of hCG is followed by increased βC-hCG immunoreactivity in urine; and (5) our results support previous studies indicating the peripheral metabolism of intact hCG to βC-hCG as the principal source for this fragment but raise the possibility that a high molecular weight-associated form, probably bound to a specific protein, may be produced by some tissues. Journal of Endocrinology (1992) 135, 175–188

Comparison of the facilitative roles of insulin and insulin-like growth factor I in the functional differentiation of granulosa cells: in vitro studies with the porcine model

1988 ◽  
Vol 117 (2) ◽  
pp. 230-240 ◽  
Author(s):  
Takeshi Maruo ◽  
Masato Hayashi ◽  
Hiroya Matsuo ◽  
Yasuo Ueda ◽  
Hajime Morikawa ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Binding Sites ◽  
Physiological Concentration ◽  
Physiological Level ◽  
Estradiol Secretion ◽  
Porcine Granulosa Cells ◽  
Igf I ◽  
Hcg Receptor

Abstract. The facilitative effects of insulin and IGF-I were compared in vitro with regard to induction of differentiated functions of porcine granulosa cells. The monolayers were maintained under serum-free conditions in the absence or presence of porcine FSH (20 μg/l), with or without graded doses of insulin or IGF-I. Concurrent treatment with IGF-I and FSH produced morphological differentiation and augmented LH/hCG receptor binding together with an enhancement in progesterone and estradiol secretion relative to treatment with FSH alone. IGF-I alone was incapable of exhibiting these effects. Insulin synergized with FSH to facilitate the granulosa cell functions except estradiol secretion. Maximal effective dose of IGF-I was 100 μg/l which is within the physiological concentration in vivo, whereas that of insulin was 1.0 mg/l, which is 1000-fold higher than the physiological level. Although the maximal effective doses of IGF-I and insulin produced a comparable increment in progesterone secretion and LH/hCG receptor induction, combined treatment with IGF-I and insulin did not prove additive. [125I]IGF-I binding revealed that specific IGF-I receptors with two classes of binding sites are present on porcine granulosa cells. No distinct differences were detected between IGF-I receptors of granulosa cells from small, medium and large follicles. Insulin was approximately 100-fold less active than IGF-I in competing for [125I]IGF-I binding. These findings suggest that porcine granulosa cells possess specific IGF-I binding sites which may mediate the cytodifferentiative actions of insulin-like peptides. Since IGF-I is more potent than insulin in amplifying the actions of FSH and maximally exerts the cytodifferentiative effects at the physiological concentration, it is likely that IGF-I plays the more important role in granulosa cell differentiation in synergy with FSH.

scholarly journals Incorporation of mannose 6-phosphate receptors into liposomes. Receptor topography and binding of α-mannosidase

1983 ◽  
Vol 214 (2) ◽  
pp. 413-419 ◽  
Author(s):  
C H Campbell ◽  
A L Miller ◽  
L H Rome
Keyword(s):  
Electron Microscopy ◽  
Bovine Serum Albumin ◽  
Binding Sites ◽  
Bovine Liver ◽  
Binding Activity ◽  
Liposome Membrane ◽  
Receptor Binding Activity ◽  
Microsomal Membranes ◽  
Cryptic Binding Sites ◽  
Mannose 6 Phosphate

A receptor that binds the lysosomal enzyme alpha-mannosidase via mannose 6-phosphate moieties (mannose 6-phosphate receptor) was purified from Swarm-rat chondrosarcoma and bovine liver microsomal membranes. Receptor-reconstituted liposomes were prepared by dialysis of taurodeoxycholate-dispersed lipids with purified mannose 6-phosphate receptor. Liposomes appeared by electron microscopy as 60-120 nm unilamellar vesicles. Receptor-reconstituted liposomes retained the ability to bind alpha-mannosidase specifically. Binding was saturable with an apparent Kd of 1 nM and was competitively inhibited by mannose 6-phosphate (Ki 2mM). Liposomes containing entrapped 125I-bovine serum albumin were used to demonstrate that treatment with 0.045% taurodeoxycholate rendered liposomes permeable to macromolecules without solubilizing the membrane. Receptor orientation in the liposome membrane was established by measuring binding of ligand to intact and detergent-treated liposomes. Unlike coated vesicles, which contain cryptic mannose 6-phosphate receptors [Campbell, Fine, Squicciarini & Rome (1983) J. Biol. Chem. 258, 2526-2533], treatment of liposomes with detergent revealed no additional cryptic binding sites. In addition, treatment of liposomes with 0.75% trypsin abolished total receptor binding activity. The results suggest that the receptor is inserted with its binding site facing the outside of the liposome.

A HISTOCHEMICAL APPROACH TO THE STUDY OF HUMAN CHORIONIC GONADOTROPHIN RECEPTORS IN THE RAT TESTIS

1976 ◽  
Vol 81 (1) ◽  
pp. 185-197 ◽  
Author(s):  
A. Dal Lago ◽  
M. T. Rolandi ◽  
S. Galli ◽  
M. Bortolussi
Keyword(s):  
Liquid Nitrogen ◽  
Specific Binding ◽  
Interstitial Cells ◽  
Binding Activity ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Binding Reaction ◽  
Receptor Complexes ◽  
Hcg Receptor ◽  
Solubilizing Effect

ABSTRACT Standard suspensions of interstitial cells in PBS were exposed to the action of various fixatives, solvents (clearing agents), temperatures and U. V. light, in order to establish the effects of such chemical and physical agents on the HCG receptors. After exposure to the various agents, the interstitial cells were incubated with [125I]HCG for 2 h at 37°C. To check the specificity of the reaction, competitive tests were performed with added excess non-iodinated HCG. Only formaldehyde fixation for short periods of time, preserved satisfactorily the specific binding activity of the receptors. A different degree of thermolability of the receptors was demonstrated, in relation to 37, 45, 54 and 60°C, while freezing in liquid nitrogen had no effect on the receptors binding activity. After the binding reaction, solvents had a significant solubilizing effect on the HCG-receptor complexes. U. V. light had no significant damaging effect on the receptors. The application of the results for a histochemical approach to the study of the HCG receptors is discussed.

Butyryl- and acetylcholine-esterases in human IVF-derived follicular fluid and luteinizing granulosa cells

2013 ◽  
Vol 121 (03) ◽  
Author(s):  
J Blohberger ◽  
D Einwang ◽  
D Berg ◽  
U Berg ◽  
S Hecht ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Human Ivf

МОДЕЛИРОВАНИЕ СИСТЕМ ЭКСТРАКОРПОРАЛЬНОГО СОЗРЕВАНИЯ ООЦИТОВ КРУПНОГО РОГАТОГО СКОТА С ИСПОЛЬЗОВАНИЕМ СТРУКТУРНЫХ КОМПОНЕНТОВ ОВАРИАЛЬНЫХ ФОЛЛИКУЛОВ *

2019 ◽  
pp. 20-22
Author(s):  
T.I. KUZMINA ◽  
I.V. CHISTYAKOVA
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Bovine Serum ◽  
Egg Quality ◽  
Fetal Bovine Serum ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cultural Systems ◽  
Fetal Bovine

Создание эффективной унифицированной системы дозревания донорских ооцитов обеспечит повышение результативности инновационных клеточных репродуктивных технологий. В исследовании проведен сравнительный мониторинг показателеймейотического созревания ооцитов коров, созревших в различных системах, дополненных структурными компонентами фолликулов (СКФ стенки фолликулов, клетки гранулезы, белки) и фолликулярной жидкостью,а также потенций к развитию из них доимплантационных эмбрионов. Анализу подверглись ооциты, прокультивированные в следующих системах:среда ТС199 с добавлением 10 фетальной бычьей сыворотки (ФБС), 50 мкг/мл эстрадиола, 10 мкг/мл лютеинизирующего гормона (ЛГ), 10 мкг/мл фолликулостимулирующего гормона (ФСГ) среда ТС199 с 10 эстральной сывороткой коров среда ТС199 с 50 жидкости из фолликулов диаметром 9 мм среда ТС199 с добавлением белков фолликулярной жидкости молекулярной массой 65 кДасреда ТС199 с 10 ФБС и 1106 клеток гранулезы среда ТС199 с 10 ФБС и тканью фолликула. В культуральные среды ко всем исследованным группам ооцитов добавляли антибиотики. Использование CКФ обеспечило значительное снижение доли ооцитов с дегенерированным хроматином, что способствовало увеличению уровня доимпланационных эмбрионов на стадии бластоцисты. Так, доля бластоцист, развившихся из ооцитов, созревших в среде со стенками фолликулов,составила43,5. В этой же группе выявлен минимальный уровень дегенерированных зародышей (6,45). Полученные данные предлагается использовать при моделировании систем дозревания ооцитов коров с целью повышения качества яйцеклеток.The creation of an effective unified maturation system of donor oocytes provides an increase in the efficiency of innovative cellular reproductive technologies. The comparative analysis of the meiotic maturation indicators of bovine oocytes, which were matured in different cultural systems modified by follicular structural components (FSC follicular walls, granulosa cells, proteins) and follicular fluid, as well as the potential for preimplantation embryonic development were evaluated in this study. Oocytes matured in following cultural systems: medium TC199 supplemented with 10 fetal bovine serum and 50 g/ml of estradiol, 10 g/ml of luteinizing hormone (LH), 10 g/ml of folliclestimulating hormone (FSH) medium TC199 with 10 estrous cow serum medium TC199 with 50 liquid from follicles with a diameter of 9 mm medium TC199 supplemented with the follicular fluid proteins with molecular weight 65 kDa medium TC199 with 10 fetal bovine serum and 1106 granulosa cells medium TC199 with the addition of 10 fetal bovine serum and follicle tissues were analyzed. Antibiotics were added to cultural media of all experimental groups of oocytes. The usage of FSC ensured the decrease in the proportion of oocytes with degenerated chromatin, which contribute the rise of the level of preimplantation embryos at the blastocyst stage. Thus, the proportion of blastocysts developed from oocytes matured in medium supplemented with follicular walls was 43.5. In the same experimental group, the number of degenerated embryos was 6.45. The obtained data are supposed to be used for modeling the cultural systems of cow oocytes in order to improve the egg quality.

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