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

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

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

Journal of Endocrinology ◽

10.1677/joe.0.1360371 ◽

1993 ◽

Vol 136 (3) ◽

pp. 371-380 ◽

Cited By ~ 2

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

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

Journal of Endocrinology ◽

10.1677/joe.0.1160307 ◽

1988 ◽

Vol 116 (2) ◽

pp. 307-312 ◽

Cited By ~ 7

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

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

Journal of Endocrinology ◽

10.1677/joe.0.1170341 ◽

1988 ◽

Vol 117 (3) ◽

pp. 341-354 ◽

Cited By ~ 12

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

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Luteotrophic effects of relaxin, chorionic gonadotrophin and FSH in common marmoset monkeys (Callithrix jacchus)

Reproduction ◽

10.1530/rep-09-0257 ◽

2010 ◽

Vol 139 (5) ◽

pp. 923-930 ◽

Cited By ~ 5

Author(s):

Nicola Beindorff ◽

Almuth Einspanier

Keyword(s):

Early Pregnancy ◽

Luteal Phase ◽

Common Marmoset ◽

Chorionic Gonadotrophin ◽

Traditional Role ◽

Local Effects ◽

Ringer’S Solution ◽

Late Luteal Phase ◽

Marmoset Monkeys

In early pregnant primates, relaxin (RLX) is highly upregulated within the corpus luteum (CL), suggesting that RLX may have an important role in the implantation of the blastocyst. Therefore, the aim of the present study was to investigate the local effects of RLX and gonadotrophins on the maintenance of the CL using anin vitromicrodialysis system. CLs of common marmoset monkeys were collected by luteectomy during different stages of the luteal phase and early pregnancy. Each CL was perfused with either Ringer's solution alone or Ringer's solution supplemented with either porcine RLX (250, 500 and 1000 ng/ml) or gonadotrophins (50 IU/ml). Application of RLX provoked a significant luteal response of progesterone (P4) and oestradiol (E2) secretions during the mid-luteal phase (500 ng/ml: P454±42%, E224±11%; 1000 ng/ml: E216±13%), and especially during the late luteal phase (250 ng/ml: P453±10%; 500 ng/ml: P444±15%; 1000 ng/ml: P462±15%, E218±7%). The effects of RLX on steroid secretion were irrespective of the RLX dosages. While treatment with human chorionic gonadotrophin did not affect luteal steroid or RLX secretion, the application of FSH resulted in a significant increase in the secretion of both P4(20±8%) and E2(37±28%), and a prominent rise in RLX during early pregnancy. In conclusion, our results demonstrate that RLX and FSH have a luteotrophic function in the marmoset monkeys; moreover, FSH has a function beyond its traditional role just as a follicle-stimulating hormone.

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

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/1764929 ◽

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.

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Control of Steroidogenesis in Small and Large Bovine Luteal. Cells

Australian Journal of Biological Sciences ◽

10.1071/bi9870331 ◽

1987 ◽

Vol 40 (3) ◽

pp. 331 ◽

Cited By ~ 45

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.

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Subcellular localization of oxytocin in the ovine corpus luteum

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y85-056 ◽

1985 ◽

Vol 63 (4) ◽

pp. 309-314 ◽

Cited By ~ 8

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.

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A comparative study of the corpus luteum

Reproduction Fertility and Development ◽

10.1071/rd9950303 ◽

1995 ◽

Vol 7 (3) ◽

pp. 303 ◽

Cited By ~ 26

Author(s):

RT Gemmell

Keyword(s):

Corpus Luteum ◽

Luteal Phase ◽

Secretory Granules ◽

Hormonal Control ◽

Egg Laying ◽

Oestrous Cycle ◽

Corpora Lutea ◽

Embryonic Diapause ◽

Alternative Reproductive Strategy ◽

Uterine Development

The corpus luteum (CL) is a transitory organ which has a regulatory role in reproduction. Sharks, amphibians and reptiles have corpora lutea that produce progesterone which influences the rate of embryonic development. The egg-laying monotremes and the two major mammalian groups, eutherian and marsupial, have a CL that secretes progesterone. Most eutherians have allowed for the uterine development of their young by extending the length of the oestrous cycle and the CL or placenta actively secretes progesterone until birth. Gestation in the marsupial does not extend beyond the length of an oestrous cycle and the major part of fetal development takes place in the pouch. Where the extension of the post-luteal phase in the eutherian has allowed for the uterine development of young, the marsupial has extended the pre-luteal phase of the oestrous cycle and has evolved an alternative reproductive strategy, embryonic diapause. The mechanism for the secretion of hormones from the CL has been controversial for many years. Densely-staining secretory granules have been observed in the CL of sharks, marsupials and eutherians. These granules have been reported to contain relaxin, oxytocin or mesotocin, and progesterone. A hypothesis to suit all available data is that all hormones secreted by the CL are transported within such granules. In conclusion, although there are obvious differences in the mode of reproduction in the two main mammalian groups, it is apparent that there is a great deal of similarity in the hormonal control of regression of the CL and parturition.

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Human chorionic gonadotropin-induced amphiregulin stimulates aromatase expression in human granulosa-lutein cells: a mechanism for estradiol production in the luteal phase

Human Reproduction ◽

10.1093/humrep/dez171 ◽

2019 ◽

Vol 34 (10) ◽

pp. 2018-2026 ◽

Cited By ~ 5

Author(s):

Lanlan Fang ◽

Yiping Yu ◽

Yiran Li ◽

Sijia Wang ◽

Ruizhe Zhang ◽

...

Keyword(s):

Menstrual Cycle ◽

Follicular Fluid ◽

Natural Science ◽

Luteal Phase ◽

Small Sample ◽

Luteal Cell ◽

Aromatase Expression ◽

Protein Levels

Abstract STUDY QUESTION Does amphiregulin (AREG), the most abundant and important epidermal growth factor receptor (EGFR) ligand in the follicular fluid, regulate aromatase expression in human granulosa-lutein (hGL) cells? SUMMARY ANSWER AREG mediates the hCG-induced up-regulation of aromatase expression and estradiol (E2) production in hGL cells. WHAT IS KNOWN ALREADY AREG expression and secretion are rapidly induced by hCG in hGL cells and mediate physiological functions of LH/hCG in the ovary. EGFR protein is expressed in follicles not only in the pre-ovulatory phase but also throughout the luteal phase of the menstrual cycle. After the LH surge, the human corpus luteum secretes high levels of E2, which regulates various luteal cell functions. Aromatase is an enzyme responsible for a key step in the biosynthesis of E2. However, whether AREG regulates aromatase expression and E2 production in hGL cells remains unexplored. STUDY DESIGN, SIZE, DURATION This study is an experimental study performed over a 1-year period. In vitro investigations examined the role of AREG in the regulation of aromatase expression and E2 production in primary hGL cells. PARTICIPANTS/MATERIALS, SETTING, METHODS Primary hGL cells were obtained from women undergoing IVF treatment in an academic research center. Aromatase mRNA and protein levels were examined after exposure of hGL cells to recombinant human AREG, hCG or LH. The EGFR tyrosine kinase inhibitor AG1478, PI3K inhibitor LY294002 and siRNAs targeting EGFR, LH receptor, StAR and AREG were used to verify the specificity of the effects and to investigate the underlying molecular mechanisms. Reverse transcription quantitative real-time PCR (RT-qPCR) and western blot were used to measure the specific mRNA and protein levels, respectively. Follicular fluid and serum were collected from 65 infertile women during IVF treatment. Pearson’s correlation analysis was performed to examine the correlation coefficient between two values. MAIN RESULTS AND THE ROLE OF CHANCE Treatment of hGL cells with AREG-stimulated aromatase expression and E2 production. Using pharmacological inhibitors and specific siRNAs, we revealed that AREG-stimulated aromatase expression and E2 production via EGFR-mediated activation of the protein kinase B (AKT) signaling pathway. In addition, inhibition of EGFR activity and AREG knockdown attenuated hCG-induced up-regulation of aromatase expression and E2 production. Importantly, the protein levels of AREG in the follicular fluid were positively correlated with the E2 levels in serum after 2 days of oocyte pick-up and in the follicular fluid of IVF patients. LARGE-SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION The in vitro setting of this study is a limitation that may not reflect the real intra-ovarian microenvironment. Clinical data were obtained from a small sample size. WIDER IMPLICATIONS OF THE FINDINGS Our results provide the first evidence that hCG-induced AREG contributes to aromatase expression and E2 production in the luteal phase of the menstrual cycle. A better understanding of the hormonal regulation of female reproductive function may help to develop new strategies for the treatment of clinical infertility. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China for Young Scientists (81601253), the specific fund of clinical medical research of Chinese Medical Association (16020160632) and the Foundation from the First Affiliated Hospital of Zhengzhou University for Young Scientists to Lanlan Fang. This work was also supported by an operating grant from the National Natural Science Foundation of China (81820108016) to Ying-Pu Sun. All authors declare no conflict of interest.

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