Specificity studies of particulate binding sites for steroid hormones in subcellular fractions of the porcine corpus luteum

1993 ◽  
Vol 136 (3) ◽  
pp. 371-380 ◽  
Author(s):  
T. A. Bramley ◽  
G. S. Menzies
Keyword(s):  
Corpus Luteum ◽  
Binding Sites ◽  
Steroid Receptors ◽  
Buoyant Density ◽  
Particulate Fraction ◽  
Luteal Cell ◽  
Similar Extent ◽  
Oestrone Sulphate ◽  
Porcine Granulosa Cell ◽  
Lipophilic Substances

ABSTRACT We have studied the binding of a number of radiolabelled steroids and lipophilic substances to porcine corpus luteum (CL) particulate fractions. Following preincubation of CL homogenates with radiolabelled progesterone or pregnenolone prior to fractionation on continuous sucrose density gradients, a broad peak of binding was observed associated with a particulate fraction of buoyant density 1·05–1·10 g/cm3. Progesterone content also peaked at a similar buoyant density (1·06–1·12 g/cm3). Pretreatment of luteal homogenates with digitonin perturbed the buoyant density of the progesterone-binding particulate fraction to 1·10–1·14 g/cm3 and sharpened the binding peak. Progesterone content was also perturbed to a similar extent by digitonin pretreatment, without release of the steroid. Oestrogens were also sequestered by this fraction, but steroid precursors (cholesterol, cholesterol ester), corticosteroids (cortisol, corticosterone), sterol conjugates (oestrone sulphate, pregnanediol glucuronide) and other lipophilic substances (arachidonic acid, phospholipid, prostaglandins E1, E2 and F2α) were not bound. Androgens were bound weakly by fractions from control gradients but, in the presence of digitonin, significant binding could be demonstrated. Radiolabelled steroids were shown to interact directly with luteal membrane fractions, rather than interacting first with cytosolic steroid receptors which then bound to membranes. Furthermore, [3H]progesterone was not bound by porcine granulosa cell particulate fractions. These observations suggest that this fraction may be involved in sequestration or packaging of progesterone for secretion by the luteal cell. Journal of Endocrinology (1993) 136, 371–380

Specific binding sites for progesterone in subcellular fractions of the porcine corpus luteum

1994 ◽  
Vol 142 (1) ◽  
pp. 101-110 ◽  
Author(s):  
G S Menzies ◽  
T A Bramley
Keyword(s):  
Corpus Luteum ◽  
Early Pregnancy ◽  
Binding Sites ◽  
Luteal Phase ◽  
Secretory Granules ◽  
Buoyant Density ◽  
Subcellular Fractions ◽  
Particulate Fraction ◽  
Luteal Cell

Abstract Subcellular fractionation of porcine corpus luteum (CL) homogenates on continuous sucrose gradients has previously demonstrated that most of the endogenous progesterone of the CL was associated with a unique particulate fraction. Exogenous radiolabelled steroids were also sequestered with some specificity by this fraction. We now report that this particulate fraction is capable of binding high levels of exogenous 3H-labelled progesterone (and pregnenolone) in vitro, but only in the presence of the saponin, digitonin. Binding was dependent on the pH, temperature and duration of incubation, and showed specificity and high affinity for progesterone (Kd, 79 nm). Androgens, oestrogens and pregnenolone competed for porcine luteal [3H] progesterone binding sites, but only at much higher concentrations, whereas cholesterol, a number of progesterone receptor agonist and antagonist analogues and inhibitors of 3β-hydroxysteroid dehydrogenase and C17-hydroxylase/C17,20-lyase did not compete. Analysis of profiles for a number of luteal cell-surface membrane and intracellular organelle markers confirmed previous studies showing the association of an NADH-cytochrome C reductase with this fraction. Moreover, the content of endogenous progesterone associated with particulate subcellular fractions isolated from porcine granulosa cell (GC) and CL homogenates at different stages of the luteal phase and early pregnancy waxed and waned with the stage of the luteal phase (and the secretory activity of the CL). Binding of [3H]progesterone in vitro equilibrated at the same buoyant density as endogenous progesterone: levels of both were highest during the mid-luteal phase and during early pregnancy, lower in early and late luteal CL, and undetectable in corpora albicantia. In contrast, relaxin secretory granules were readily resolved from progesterone binding sites. We propose that these particulate progesterone binding sites may be involved in the sequestration and/or packaging of newly-synthesized steroid for secretion by the luteal cell, or may mediate actions of progesterone within the luteal cell. Journal of Endocrinology (1994) 142, 101–110

Association of progesterone with a unique particulate fraction of the human corpus luteum

1988 ◽  
Vol 116 (2) ◽  
pp. 307-312 ◽  
Author(s):  
T. A. Bramley ◽  
G. S. Menzies
Keyword(s):  
Plasma Membrane ◽  
Corpus Luteum ◽  
Plasma Membranes ◽  
Buoyant Density ◽  
Particulate Fraction ◽  
Luteal Cell ◽  
Cell Plasma Membrane ◽  
Intracellular Organelle ◽  
Cell Plasma ◽  
Human Corpus Luteum

ABSTRACT Homogenates of human corpus luteum were fractionated on continuous sucrose density gradients, with and without pretreatment with digitonin to perturb plasma membranes. Fractions of each gradient were assayed for steroid content and a range of plasma membrane and intracellular organelle markers. Progesterone and oestradiol were associated with a particulate fraction (buoyant density, 1·08–1·13 g/cm3). The buoyant density distribution of these steroids was distinct from those of the luteal cell plasma membrane and intracellular organelle markers tested. Treatment with digitonin increased the buoyant density of both progesterone and oestradiol. If steroids are contained in distinct vesicles, these vesicles may be involved in the sequestration of newly synthesized steroid and its movement to the cell surface for release into the circulation. J. Endocr. (1988) 116, 307–312

Subcellular fractionation of the porcine corpus luteum: sequestration of progesterone in a unique particulate fraction

1988 ◽  
Vol 117 (3) ◽  
pp. 341-354 ◽  
Author(s):  
T. A. Bramley ◽  
G. S. Menzies
Keyword(s):  
Cytochrome C ◽  
Cell Surface ◽  
Corpus Luteum ◽  
Reductase Activity ◽  
Buoyant Density ◽  
Particulate Fraction ◽  
Luteal Cell ◽  
Cytochrome C Reductase ◽  
Intracellular Organelles ◽  
Nadh Cytochrome

ABSTRACT Homogenates of porcine corpus luteum were subjected to fractionation by differential-rate centrifugation or sucrose density gradient fractionation, with or without pretreatment with digitonin. Fractions of each gradient were assayed for a number of markers characteristic of the major intracellular organelles and cell-surface membranes, and for progesterone content. The majority of the progesterone content of homogenates of porcine corpus luteum was associated with a low-density particulate fraction which equilibrated at a buoyant density of 1·07–1·09 g/cm3. Pretreatment with digitonin increased the buoyant density of the progesterone-enriched fraction markedly (to 1·13–1·15 g/cm3) without causing release of steroid. The density distributions of progesterone content in control and digitonin-treated luteal gradient fractions were quite distinct from those of the major intracellular organelles and luteal cell-surface membranes. However, NADH–cytochrome C reductase activity (but not other endoplasmic reticulum markers) was also enriched in this fraction. The results suggest that most of the progesterone of the porcine corpus luteum is associated with a unique particulate fraction which is enriched in digitonin-reactive lipids and NADH–cytochrome C reductase activity. J. Endocr. (1988) 117, 341–354

scholarly journals Differential distribution of gelatinases and tissue inhibitor of metalloproteinase-1 in the rat ovary

1998 ◽  
Vol 158 (2) ◽  
pp. 221-228 ◽  
Author(s):  
P Bagavandoss
Keyword(s):  
Plasma Membrane ◽  
Corpus Luteum ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Neonatal Rat ◽  
Luteal Cell ◽  
Surface Epithelium ◽  
Tissue Inhibitor ◽  
Differential Distribution ◽  
Thecal Cells ◽  
Rat Ovary

The distribution of gelatinases/matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in neonatal and gonadotropin-primed immature rat ovaries was studied by immunofluorescent microscopy. Immature female Long-Evans rats were primed with 15 IU pregnant mare's serum gonadotropin (PMSG) in 100 microliters PBS. Two days later, to induce ovulation, the rats were injected with human chorionic gonadotropin (hCG, 5 IU/100 microliters PBS). The animals were killed at appropriate times and the ovaries removed and processed for cryostat or paraffin sectioning. Ovaries were also obtained from 7-day-old neonatal rats and processed as above. In the neonatal rat ovary, MMP-2 was present in the follicle and in the ovarian surface epithelium. MMP-9 was not detectable in the neonatal ovary. TIMP-1 was present in the oocyte and in the surface epithelium. In the PMSG-primed ovary, MMP-2 was present in the granulosa and thecal cells of the ovary. MMP-9 distribution, however, was restricted to the interstitial and thecal cells. TIMP-1 was mainly present in the blood vessels and thecal cells, with minor staining in the granulosa cells. In the developing corpus luteum, luteal and endothelial cells were positive for MMP-2. MMP-9 localization was restricted to the plasma membrane of the luteal and interstitial cells. TIMP-1 was clearly observed in the luteal capillaries and, to a lesser extent, in the luteal cell plasma membrane. This distribution of MMP-2, MMP-9, and TIMP-1 in the corpus luteum persisted throughout the life span of the corpus luteum. The spatial and temporal distribution of the gelatinases and TIMP-1 suggests unique roles for these proteins in the rat ovary.

scholarly journals HIF-1α Activation Promotes Luteolysis by Enhancing ROS Levels in the Corpus Luteum of Pseudopregnant Rats

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Zonghao Tang ◽  
Jiajie Chen ◽  
Zhenghong Zhang ◽  
Jingjing Bi ◽  
Renfeng Xu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Corpus Luteum ◽  
Cell Apoptosis ◽  
In Vitro Study ◽  
Luteal Cell ◽  
Independent Manner ◽  
Luteal Regression ◽  
Luteal Cells

The increase of oxidative stress is one of the important characteristics of mammalian luteal regression. Previous investigations have revealed the essential role of reactive oxygen species (ROS) in luteal cell death during luteolysis, while it is unknown how ROS is regulated in this process. Considering the decrease of blood flow and increase of PGF2α during luteolysis, we hypothesized that the HIF-1α pathway may be involved in the regulation of ROS in the luteal cell of the late corpus luteum (CL). Here, by using a pseudopregnant rat model, we showed that the level of both HIF-1α and its downstream BNIP3 was increased during luteal regression. Consistently, we observed the increase of autophagy level during luteolysis, which is regulated in a Beclin1-independent manner. Comparing with early (Day 7 of pseudopregnancy) and middle CL (Day 14), the level of ROS was significantly increased in late CL, indicating the contribution of oxidative stress in luteolysis. Inhibition of HIF-1α by echinomycin (Ech), a potent HIF-1α inhibitor, ameliorated the upregulation of BNIP3 and NIX, as well as the induction of autophagy and the accumulation of ROS in luteal cells on Day 21 of pseudopregnancy. Morphologically, Ech treatment delayed the atrophy of the luteal structure at the late-luteal stage. An in vitro study indicated that inhibition of HIF-1α can also attenuate PGF2α-induced ROS and luteal cell apoptosis. Furthermore, the decrease of cell apoptosis can also be observed by ROS inhibition under PGF2α treatment. Taken together, our results indicated that HIF-1α signaling is involved in the regression of CL by modulating ROS production via orchestrating autophagy. Inhibition of HIF-1α could obviously hamper the apoptosis of luteal cells and the process of luteal regression.

scholarly journals Control of Steroidogenesis in Small and Large Bovine Luteal. Cells

1987 ◽  
Vol 40 (3) ◽  
pp. 331 ◽  
Author(s):  
William Hansel ◽  
Hector W Alila ◽  
Joseph P Dowd ◽  
Xiangzhong Yang
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Corpus Luteum ◽  
Luteal Cell ◽  
Lipoxygenase Pathway ◽  
Luteal Cells ◽  
Kinase C ◽  
Bovine Corpus Luteum ◽  
Progesterone Synthesis

Evidence was cited to show that: (1) prostacyclin (PGI2) plays a luteotrophic role in the bovine corpus luteum and that products of the lipoxygenase pathway of arachidonic acid metabolism, especially 5-hydroxyeicosatetraenoic acid play luteolytic roles; (2) oxytocin of luteal cell origin plays a role in development, and possibly in regression, of the bovine corpus luteum; and (3) luteal cells arise from two sources; the characteristic small luteal cells at all stages of the o~strous cycle and pregnancy are of theca cell origin; the large cells are of granulosa cell origin early in the cycle, but a population of theca-derived large cells appears later in the cycle. Results of in vitro studies with total dispersed cells and essentially pure preparations of large and small luteal cells indicate that : (1) the recently described Ca2+ -polyphosphoinositol-protein kinase C second messenger system is involved in progesterone synthesis in the bovine corpus luteum; (2) activation of protein kinase C is stimulatory to progesterone synthesis in the small luteal cells; (3) activation of protein kinase C has no effect on progesterone synthesis in the large luteal cells; and (4) protein kinase C exerts its luteotrophic effect in total cell preparations, in part at least, by stimulating the production of prostacyclin. The protein kinase C system may cause down regulation of LH receptors in the large cells.

Subcellular localization of oxytocin in the ovine corpus luteum

1985 ◽  
Vol 63 (4) ◽  
pp. 309-314 ◽  
Author(s):  
G. E. Rice ◽  
G. D. Thorburn
Keyword(s):  
Subcellular Localization ◽  
Physicochemical Properties ◽  
Corpus Luteum ◽  
Density Gradient Centrifugation ◽  
Secretory Granules ◽  
Gradient Centrifugation ◽  
Particulate Fraction ◽  
Dense Granules ◽  
Oxytocin Release ◽  
Midluteal Phase

The subcellular localization of oxytocin within the corpus luteum of sheep was investigated using differential and density gradient centrifugation. Oxytocin was associated with a particulate fraction which sedimented to a density of 1.054 – 1.061 g/mL. The exclusion of [3H]oxytocin from this particulate fraction is indicative that particulate oxytocin represents endogenous compartmentalization. Particulate oxytocin, incubated in buffered medium at 37 °C, was stable for up to 1 h and the release of oxytocin was not affected by the pH of the incubation medium, over the range 5.5 – 8.5. Oxytocin release, however, was stimulated by incubating particle-bound oxytocin in buffered medium of low osmolality (<200 mosmol). These data are similar to the physicochemical properties reported for peptide-containing neurohypophysial secretory granules. Ultrastructural analysis of oxytocin-containing fractions revealed the presence of electron-dense granules (diameter, 200–250 nm). These data are suggestive that oxytocin, in the corpus luteum of sheep, is contained within a population of secretory granules which occur in high numbers during the midluteal phase of the oestrous cycle.

scholarly journals Association of luteal cell degeneration and progesterone deficiency with lysosomal storage disorder mucolipidosis type IV in Mcoln1−/− mouse model†

2019 ◽  
Vol 101 (4) ◽  
pp. 782-790 ◽  
Author(s):  
Zidao Wang ◽  
Ahmed E El Zowalaty ◽  
Yuehuan Li ◽  
Christian L Andersen ◽  
Xiaoqin Ye
Keyword(s):  
Corpus Luteum ◽  
Lysosomal Storage Disorder ◽  
Luteal Cell ◽  
Lysosomal Storage ◽  
Cell Degeneration ◽  
Mucolipidosis Type Iv ◽  
Type Iv ◽  
Mitochondrial Functions ◽  
Storage Disorder ◽  
Mucolipidosis Type

Abstract Transient receptor potential cation channel, mucolipin subfamily, member 1 (TRPML1) (MCOLN1/Mcoln1) is a lysosomal counter ion channel. Mutations in MCOLN1 cause mucolipidosis type IV (MLIV), a progressive and severe lysosomal storage disorder with a slow onset. Mcoln1−/− mice recapitulate typical MLIV phenotypes but roles of TRPML1 in female reproduction are unknown. Despite normal mating activities, Mcoln1−/− female mice had reduced fertility at 2 months old and quickly became infertile at 5 months old. Progesterone deficiency was detected on 4.5 days post coitum/gestation day 4.5 (D4.5). Immunohistochemistry revealed TRPML1 expression in luteal cells of wild type corpus luteum (CL). Corpus luteum formation was not impaired in 5–6 months old Mcoln1−/− females indicated by comparable CL numbers in control and Mcoln1−/− ovaries on both D1.5 and D4.5. In the 5–6 months old Mcoln1−/− ovaries, histology revealed less defined corpus luteal cord formation, extensive luteal cell vacuolization and degeneration; immunofluorescence revealed disorganized staining of collagen IV, a basal lamina marker for endothelial cells; Nile Red staining detected lipid droplet accumulation, a typical phenotype of MLIV; immunofluorescence of heat shock protein 60 (HSP60, a mitochondrial marker) and in situ hybridization of steroidogenic acute regulatory protein (StAR, for the rate-limiting step of steroidogenesis) showed reduced expression of HSP60 and StAR, indicating impaired mitochondrial functions. Luteal cell degeneration and impaired mitochondrial functions can both contribute to progesterone deficiency in the Mcoln1−/− mice. This study demonstrates a novel function of TRPML1 in maintaining CL luteal cell integrity and function.

Hormonal dependency of cerebral meningiomas

1990 ◽  
Vol 73 (5) ◽  
pp. 743-749 ◽  
Author(s):  
Uwe M. H. Schrell ◽  
Eric F. Adams ◽  
Rudolf Fahlbusch ◽  
Robert Greb ◽  
Gustav Jirikowski ◽  
...  
Keyword(s):  
Estrogen Receptors ◽  
Binding Sites ◽  
Solid Phase ◽  
Steroid Receptors ◽  
Progesterone Receptors ◽  
Nuclear Compartment ◽  
Content Type ◽  
High Affinity ◽  
Immunohistochemical Techniques ◽  
Fine Print

✓ Female sex steroid receptors were examined in 50 human cerebral meningiomas. For estrogen receptors, high-affinity binding sites (dissociation constant (Kd): 0.05 to 0.2 nM) were found in the cytosolic fraction with a capacity of less than 4 fmol/mg protein in 10 meningiomas using a dextran-coated charcoal (DCC) assay. In the same cytosolic fraction, the solid-phase enzyme immunoassay revealed only one cytosol with a positive colorimetric reaction equal to 5 fmol/mg protein. However, in the nuclear compartment, none of the tumors stained positively for estrogen receptors with immunohistochemical techniques. In addition, the most convincing evidence for the absence of estrogen receptors was obtained by in situ hybridization using an oligonucleotide probe complementary to a fraction of the human receptor messenger ribonucleic acid (mRNA). In none of the 50 meningiomas was the expression of estrogen mRNA coding for the estrogen receptor detected. For progesterone receptors, high-affinity binding sites (Kd: 0.3 to 2.6 nM) were found in 49 of the 50 tumors using a DCC assay. In the same cytosols, solid-phase enzyme immunoassay revealed that each tumor was positive for progesterone receptors. However, in the nuclear compartment, only five tumors had partially positive staining for progesterone receptors with immunohistochemical techniques. Within the confines of this study, it is concluded that: 1) the estrogen receptor is generally absent in meningioma tissue, and 2) the progesterone receptor is mainly absent in the nuclear compartment, leading to the conclusion that the cytosolic progesterone receptor may be an inactive form. This study suggests that female sex steroid receptors are not primarily involved in the proliferative rate of cerebral meningiomas and that they are of no current significance as markers for adjuvant medical therapy of most meningiomas.

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