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Results for “Luteinization”

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PubMed1989

Effect of indomethacin on the vascular architecture of preovulatory ovine follicles: possible implication in the luteinized unruptured follicle syndrome.

Ovarian follicles sometimes fail to rupture and accumulate large quantities of fluid, yet undergo luteinization and form a steroidogenically competent luteal structure, the luteinized unruptured follicle syndrome. This condition can be mimicked in mammals by administration of indomethacin, an inhibitor of biosynthesis of prostaglandins. Blockade of ovulation by this drug is exerted at the follicular level. Ovulatory failure in sheep given a single intramuscular injection of indomethacin after induction of a surge of of luteinizing hormone was associated with follicular hyperemia as assessed by scanning electron microscopic examination of microcorrosion vascular casts and light microscopic quantification of follicular vascular space. The apical stigma of control (ovulatory) follicles was ischemic. The luteinized unruptured follicle syndrome appears to be the consequence of a chronic follicular inflammatory-like reaction involving inhibition of synthesis of prostaglandins.

Animals↗

PubMed1997

Immunodetection of cotinine protein in granulosa-lutein cells of women exposed to cigarette smoke.

OBJECTIVE: To detect immunoreactivity to cotinine protein, a major metabolite of nicotine, in granulosa-lutein cells from patients exposed to cigarette smoke, as measured by levels of cotinine in follicular fluid (FF) samples. DESIGN: Controlled immunocytochemical study. SETTING: Hospital IVF-ET program treating infertile patients. PATIENT(S): Twenty-eight women classified by self-reported smoking habits: active smokers (n = 17), passive smokers (n = 4), and nonsmokers (n = 7). INTERVENTION(S): Ovarian hyperstimulation. MAIN OUTCOME MEASURE(S): Grades of immunostaining intensity were assessed in granulosa-lutein cells. Patient scores of cell immunostaining were calculated and regressed on levels of FF cotinine. RESULT(S): Cotinine levels in FF were higher in active smokers than in passive smokers or nonsmokers. Cotinine immunostaining was visualized in the nucleus and cytoplasm of granulosa-lutein cells. Mean grades and mean scores of immunostaining intensity were higher in active smokers than in passive smokers or nonsmokers. There was a strong positive correlation between scores of cell immunostaining and FF cotinine levels. CONCLUSION(S): The association between cotinine expression in granulosa-lutein cells and FF cotinine provides reliable evidence for a dose-related effect. This constituent of cigarette smoke appears to interact directly with and incorporate into these ovarian cells. Our approach seems useful for monitoring ovarian exposure to environmental toxins.

Animals↗

PubMed1997

Sequence of the bovine HDL-receptor (SR-BI) cDNA and changes in receptor mRNA expression during granulosa cell luteinization in vivo and in vitro.

Steroidogenic activity in the mature corpus luteum of most mammals depends upon provision of cholesterol from the circulating lipoproteins. In cattle, as in many species, high-density lipoprotein (HDL) is the major lipoprotein involved. The recent identification of the scavenger receptor SR-BI as an HDL-receptor allows control of this process to be investigated more closely. In this study, we have sequenced the bovine SR-BI HDL-receptor and examined changes in expression of the receptor mRNA during corpus luteum development in vivo and granulosa cell luteinization in vitro. Sequencing of the bovine HDL-receptor showed that it codes for a protein of 509 amino acids with close identity to hamster, mouse, rat and human sequences. Examination of the tissue distribution of the HDL-receptor mRNA showed high levels in adrenal cortex and corpus luteum and lower levels in spleen and liver. Using a semi-quantitative, reverse transcription-polymerase chain reaction technique levels of HDL-receptor mRNA were measured in corpora lutea from cattle at known stages of the oestrus cycle and in bovine granulosa cells luteinized in culture. Levels of HDL-receptor mRNA were low in isolated bovine granulosa cells, but increased 7-fold during corpus luteum development in vivo and 5-fold during granulosa cell luteinization in culture. Results show that luteinization of granulosa cells is associated with an increase in HDL-receptor RNA levels which, along with changes in steroidogenic enzyme activity, is likely to explain the marked increase in steroidogenic capacity which occurs during corpus luteum formation.

Amino Acid Sequence↗

PubMed1966

Ovarian steroid cells. I. Differentiation of the lutein cell from the granulosa follicle cell during the preovulatory stage and under the influence of exogenous gonadotrophins.

The granulosa follicle cell of the Graafian follicle of the rabbit ovary differentiates into a lutein cell involved in steroid synthesis. Cytological events which occur within the granulosa cell of the normally stimulated follicle prior to ovulation have been duplicated by the intrafollicular injection of exogenous gonadotrophin. The luteinization of the granulosa cells involves the accumulation of 250- to 300-A, electron-opaque, spherical granules, dispersed within the cytoplasmic matrix, which have been identified as glycogen with the PAS-staining procedure. Further development of the granulosa cell following ovulation involves an increase in cell size, a decrease in the number of RNP particles, and an accumulation of an abundant system of intracellular membranes (agranular endoplasmic reticulum). Glycogen granules first appear in the granulosa cells as the separate, monoparticulate form. After follicle rupture and the formation of agranular endoplasmic reticulum, glycogen particles are present in a rosette arrangement within membrane-bounded vacuoles. The rosette arrangement of glycogen particles is also found dispersed within the cytoplasmic matrix of the lutein cell during the later stages of the cell life-span. Injection of luteinizing hormone or human chorionic gonadotrophin into a mature follicle also produces a marked accumulation of monoparticulate glycogen in the majority of granulosa cells, within 30 min. Cytoplasmic extensions which contain the glycogen masses are noticeably free of RNP particles.

Animals↗

PubMed1997

Vascular endothelial growth factor production by human luteinized granulosa cells in vitro.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) originating from the follicle or corpus luteum may be a physiological regulator of ovulation and neovascularization of luteinizing tissue, as well as a pathological factor in the development of ovarian hyperstimulation syndrome (OHSS). The objective of this study was to quantify VEGF production by human luteinized granulosa cells in vitro and to determine if gonadotrophin stimulates VEGF production directly and/or indirectly via enhanced synthesis of progesterone. In study 1, luteinized granulosa cells collected from women undergoing ovarian stimulation for in-vitro fertilization were cultured in the presence and absence of human chorionic gonadotrophin (HCG; 100 ng/ml) and/or low density lipoprotein (LDL; 100 microg protein/ml). In study 2, the progesterone synthesis inhibitor trilostane (250 ng/ml) and/or a progesterone receptor antagonist ZK137.316 (3.2 microM) were also added. Medium was harvested on days 1, 3, 5, 7 and 9 of culture and assayed for VEGF and progesterone. Results of study 1 were divided into two categories based on control concentrations of VEGF on day 1: 'low producers' (n = 6; <750 pg VEGF/ml) and 'high producers' (n = 5; >1000 pg VEGF/ml; P < 0.01). VEGF concentrations in cultures of both low and high producers increased (P < 0.01) from day 1 to maximal values on day 3, then steadily declined through to day 9. Chronic exposure to LDL or HCG increased (P < 0.05) VEGF concentrations in cultures of low producers by day 3 and day 5 respectively. In contrast, LDL did not alter VEGF concentrations in cultures of high producers and HCG did not increase VEGF concentrations until day 7. Nevertheless, acute exposure to HCG beginning on day 7 increased (P < 0.05) VEGF concentrations 3-fold in cultures of low or high producers. In study 2, trilostane treatment decreased (P < 0.05) progesterone concentrations by 91% on day 1 of culture but had no effect on VEGF concentrations on any day. ZK137.316 alone or with trilostane did not affect VEGF synthesis. These results suggest that VEGF production by luteinized granulosa cells is enhanced by gonadotrophin (HCG) independent of gonadotrophin-stimulated progesterone synthesis. These data are consistent with the hypothesis that the exacerbation of OHSS in early pregnancy is mediated by the CG stimulation of luteal VEGF production.

Adult↗

PubMed1998

Steroidogenesis in luteinized granulosa cell cultures varies with follicular priming regimen.

During follicular development, a co-ordinated gonadotrophin and endocrine environment is believed to be essential for normal function of the resulting corpus luteum. Whether differences in the gonadotrophins used to promote follicular development can have lasting effects on granulosa cells after they have undergone luteinization and culture, remains to be studied. We measured steroid production under basal and human chorionic gonadotrophin (HCG) stimulation in short and long term cultures of luteinizing granulosa cells obtained from normal ovulatory women undergoing assisted folliculogenesis with either human menopausal gonadotrophin (HMG) or follicle stimulating hormone (FSH). Basal progesterone and oestradiol production by luteinized granulosa cells obtained from follicles stimulated to develop with FSH was significantly greater than that from HMG derived follicles (P < 0.001). In short term cultures, treatment with 10 IU HCG caused a 10-fold increase in progesterone release by cells from FSH stimulated follicles, whereas cells of HMG origin produced only 5-fold more progesterone (P < 0.0001). In cultures that were maintained for 2 weeks, progesterone secretion was reduced, but a similar trend in HCG responsiveness was observed. These experiments demonstrate that the composition of the gonadotrophins used to promote follicular development in vivo leads to differences in granulosa cell steroidogenesis which are evident after luteinization and culture. They additionally support the notion that the environment of follicular development will be reflected in the resulting corpus luteum.

Cells, Cultured↗

PubMed1996

Gonadotrophin-releasing hormone and triptorelin inhibit the follicle stimulating hormone-induced response in human primary cultured granulosa-lutein cells.

Cyclic AMP (c-AMP)-dependent cell shape changes can be used to study the gonadotrophin response in cultured human granulosa-lutein cells. The same approach has been developed here to determine the direct potential antigonadotrophic effect of gonadotrophin-releasing hormone (GnRH) and a GnRH agonist (triptorelin) on human granulosa-lutein cells. Treatment with triptorelin or GnRH alone for 1 h did not affect granulosa-lutein cell morphology. However, in the presence of stimulatory doses of follicle stimulating hormone (FSH), triptorelin (5 x 10(-7)-5 x 10(-6) M) and GnRH (10(-11)-10(-9) M) inhibited the FSH-induced c-AMP-dependent response. The antigonadotrophic effect of triptorelin was prevented by two GnRH antagonists, indicating that triptorelin acts via specific GnRH binding sites. On the other hand, triptorelin failed to inhibit human chorionic gonadotrophin- and forskolin-mediated morphological changes. Our results suggest that the GnRH agonist interacts specifically with the FSH-induced c-AMP-dependent cascade of events, at a site located ahead of that of c-AMP generation. In conclusion, GnRH and triptorelin strongly inhibit FSH-mediated function in human granulosa-lutein cells in culture. This inhibition might play a role in the low follicular development rates observed in some patients treated with GnRH agonist + gonadotrophins for ovarian stimulation.

Adenosine Monophosphate↗

PubMed1997

Integrin alpha5 is expressed on human luteinizing granulosa cells during corpus luteum formation, and its expression is enhanced by human chorionic gonadotrophin in vitro.

We previously reported that human granulosa cells (GC) express integrin alpha6beta1, in large follicles and early corpus luteum (CL). In this study, we examined the expression of integrin alpha5 and fibronectin (FN) by immunohistochemistry. Integrin alpha5 was undetectable on human GC in preovulatory follicles, but it was intensely expressed on luteinizing GC of CL in the early luteal phase, and it was weakly expressed on large luteal cells (LL) in the midluteal phase. FN was similarly expressed between GC/LL. Thus, integrin alpha5 and FN were stage-specifically expressed on/between luteinizing GC in the early luteal phase. Next we examined the effect of human chorionic gonadotrophin (HCG) on expression of integrin alpha5 on luteinizing GC in vitro. GC obtained from preovulatory follicles were cultured for 3 days without (control) or with HCG (1 IU/ml), and expression of integrin alpha5 was examined by flow cytometry. Although the positivity rate did not differ, the mean relative fluorescence intensity was 3.4-fold higher in the HCG-treated group (n = 8, P < 0.01). These findings indicate that integrin alpha5 is rapidly expressed on GC during luteinization, probably under LH stimulation, and suggest that integrin alpha5 is involved in CL formation via interaction with its ligand FN.

Antigens, CD↗

PubMed2003

Melanoma cell adhesion molecule (MCAM/CD146) is expressed on human luteinizing granulosa cells: enhancement of its expression by hCG, interleukin-1 and tumour necrosis factor-alpha.

Melanoma cell adhesion molecule (MCAM) was originally reported to be involved in the invasion and progression of melanoma. It was also shown to be responsible for the attachment of cells to endothelial cells. In this study, we demonstrated by immunohistochemistry that immunoreactive MCAM was not expressed on granulosa cells in the pre-ovulatory follicle, but it was clearly detected in large luteal cells in corpora lutea from the mid-luteal phase of the menstrual cycle. Northern blotting analysis confirmed the expression of MCAM mRNA in corpus luteum. MCAM was weakly detected by immunocytochemical staining in human luteinizing granulosa cells isolated from patients undergoing IVF treatment. Its expression was found to be increased during time in culture of these cells. Flow cytometry and Northern blot analysis revealed that MCAM expression on luteinizing granulosa cells was enhanced when the cells were cultured for 5 days in the presence of hCG (1 IU/ml) or cytokines such as interleukin-1alpha (10 ng/ml) and tumour necrosis factor-alpha (10 ng/ml). No significant difference of MCAM expression was observed between the cultures under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. These results indicate that luteinizing granulosa cells express MCAM and that MCAM expression is regulated by LH/hCG and cytokines during luteinization. Since MCAM has been reported to mediate cellular interaction with endothelial cells, this molecule may play a role in neovascularization during corpus luteum formation in the human ovary.

Antigens, CD↗

PubMed1991

Premature luteinization: treatment and incidence in natural cycles.

The incidence of premature luteinization was evaluated in 400 women with a history of infertility (greater than or equal to 18 months). After its diagnosis, this condition was treated with ovulation-inducing drugs in the early follicular phase in an attempt to accelerate follicular maturation before the luteinizing hormone (LH) surge. Premature luteinization was diagnosed if serum progesterone levels greater than 1.5 ng/ml were associated with an LH surge before the serum oestradiol level reached 200 pg/ml and before the follicle was mature. Fifty-two of 400 (13%) women demonstrated premature luteinization in two consecutive cycles. Fourteen of 52 (27%) women corrected the problem with a clomiphene citrate regimen, as compared with 32 of 38 (75%) treated with HMG and HCG; conception rates were 83 and 50%, respectively, for the patients who responded to the two regimens. Overall, regimens utilized in this study resulted in a 58% pregnancy rate in 6 months.

Female↗

PubMed1993

Premature luteinization as detected by elevated serum progesterone is associated with a higher pregnancy rate in donor oocyte in-vitro fertilization.

Premature luteinization has been reported to be associated with decreased pregnancy rates in patients undergoing in-vitro fertilization. However, the detrimental effect created by a pre-aspiration rise in progesterone is difficult to assess since ovarian stimulation affects both oocyte quality and endometrial receptivity. Therefore, the relationship between premature luteinization and pregnancy rates remains uncertain. To achieve improved control for confounding variables, we studied premature luteinization in ovum donors of proven fertility. A total of 114 consecutive ovum donation cycles using pituitary suppression with a gonadotrophin-releasing hormone agonist followed by gonadotrophin stimulation were examined. Serum progesterone concentration on the day of administration of human chorionic gonadotrophin (HCG) was > 1.2 ng/ml in 29% of patients. Patients were divided into two groups based on this value. There was a significant increase in clinical pregnancy rates per embryo transfer in the group with higher progesterone concentrations (53 versus 25%, P = 0.012), as well as significantly more oocytes obtained at aspiration (19.6 +/- 10.4 versus 13.3 +/- 5.4, P < 0.001), and significantly higher peak serum oestradiol values (3903 +/- 1787 versus 2453 +/- 1232 pg/ml, P < 0.001). There were no significant differences between groups due to age, degree of stimulation or the number of embryos transferred. We conclude that premature luteinization as based on elevated serum progesterone concentration is a common occurrence in oocyte donors, reflects healthy follicular development, and is associated with increased pregnancy rates.

Adult↗

PubMed1985

Discrete hypothalamic distribution of luteinizing hormone-releasing hormone (LHRH) content and of LHRH-degrading activity in laying and nonlaying hens.

Hypothalamic enzymatic luteinizing hormone-releasing hormone (LHRH)-degrading activity (LHRH-DA) may play a physiologic role in the neuroendocrine control of LHRH in mammals. The present study analyzes the existence and possible physiologic role of LHRH-DA in birds. The LHRH content in discrete hypothalamic samples of laying and nonlaying hens was correlated to their LHRH-DA. Degrading activity was assessed by high-performance liquid chromatography (HPLC) of chicken LHRH and of its degradation fragments. Luteinizing hormone-releasing hormone content was estimated by radioimmunoassay. Luteinizing hormone-releasing hormone content of discrete medial preoptic, infundibulum, and arcuate samples, as well as serum LH and progesterone levels, were higher (P less than 0.05) in laying than in nonlaying hens. The LHRH content of these hypothalamic areas was also higher (P less than 0.05) than those of immediately adjacent areas, in both animal groups. Luteinizing hormone-releasing hormone-degrading activity, which generates LHRH1-5 as the main degradation fragment, was higher (P less than 0.01) in the infundibulum of laying than in nonlaying hens. It was also higher (P less than 0.01) in samples from the infundibulum and medial preoptic area than in immediately adjacent lateral samples. Finally, LHRH-DA, showing a similar HPLC profile of degradation fragments, was also present in areas of low or undetectable LHRH content.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Androgen metabolism by porcine granulosa cells during the process of luteinization in vitro: identification of 19-oic-androstenedione as a major metabolite and possible precursor for the formation of C18 neutral steroids.

The present studies were conducted to define the pathway(s) by which androstenedione is metabolized in porcine granulosa cells (pGC) and determine whether metabolism of this steroid is affected by in vitro luteinization. pGC isolated from large preovulatory follicles were cultured for up to 2 days in the presence of 5 microM unlabeled or [4-14C]-labeled androstenedione. Metabolism of androstenedione was assessed by HPLC, using in-line liquid scintillation detection. Metabolite identification was confirmed by gas chromatography-mass spectrometry of HPLC fractions isolated from medium conditioned by granulosa cells (pGCCM) cultured for 48 h in the presence of unlabeled androstenedione. The metabolites identified were 19-oic-androstenedione (3,17-dioxo-4-androsten-19-oic acid), 19-hydroxytestosterone, 19-hydroxyandrostenedione, 19-nor-testosterone, an estrenolone of as yet unproven stereoisomerism, 5(10)-estrene-3 beta, 17 beta-diol, 17 beta-estradiol, testosterone, and 19-nor-androstenedione. Results indicate that 19-nor-androstenedione is artifactually derived from 19-oic-androstenedione as a result of degradation in storage and during isolation. After metabolite identification, studies of the time course of androstenedione metabolism by pGC during in vitro luteinization were conducted. 17 beta-Estradiol and 19-oic-androstenedione were the predominant metabolites, and accumulation of these steroids was virtually identical. Production of these metabolites was maximal during the first 12 h of culture. The accumulation of 5(10)-estrene-3 beta,17 beta-diol and 19-nor-testosterone was maximal at 48 h of culture, with 5(10)-estrene-3 beta,17 beta-diol consistently accumulating in greater concentrations than 19-nor-testosterone. Aromatase activity of pGC was negligible from 36-48 h of culture, as demonstrated by minimal accumulation of 17 beta-estradiol during this period of culture. The accumulation of 19-oic-androstenedione, 5(10)-estrene-3 beta,17 beta-diol, and 19-nor-testosterone was also negligible during this latter time period, suggesting that their formation is associated with aromatase. From these results, pGC from preovulatory follicles undergoing luteinization in vitro lose the ability to convert androstenedione to estrogens. The formation of 19-oic-androstenedione, shown here for the first time, parallels the formation of 17 beta-estradiol, and this acidic steroid is proposed to be a product of aromatase. As reported in previous studies, pGC do produce C18 neutral steroids from exogenous androstenedione. The production of these steroids requires an active aromatase to produce their immediate precursor, which is here hypothesized to be 19-oic-androstenedione. However, their maximal production does not commence until aromatase activity has declined, and it is hypothesized that their production depends on modifications in steroid metabolism associated with luteinization.

Androgens↗

PubMed1998

Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian models.

Steroidogenic cells in rats and mice obtain most of their cholesterol for steroid production and cholesteryl ester (CE) storage via the selective uptake pathway in which high density lipoprotein CE (HDL-CE) is taken into the cell without the uptake and degradation of the HDL particle. A number of recent studies show that the scavenger receptor, class B, type I (SR-BI) can mediate HDL-CE selective uptake in cultured cells and suggest that this receptor may be responsible for HDL-CE selective uptake in steroidogenic cells in vivo. In the current study we examine the relationship between SR-BI expression and HDL-CE selective uptake in the gonadotropin-primed, luteinized rat ovary and in the ovary that is desensitized by multiple gonadotropin treatments. Results from this study demonstrate a tight association between expression of SR-BI and measurements of HDL-CE selective uptake regardless of the steroidogenic state of the ovary. Thus, in the luteinized ovary (which is actively producing progestins), HDL-CE selective uptake is high, as is the expression of SR-BI. In the desensitized ovary (where CE content is reduced by 90% and progestin production is virtually absent), HDL-CE selective uptake and SR-BI are induced 2- to 3-fold compared with those in the luteinized ovary. These data argue that SR-BI can be regulated by the cholesterol status of the luteal cell independently of gonadotropic stimulation. Immunostaining at the light microscopic level showed strong expression of SR-BI specifically on the surface of luteal cells in the luteinized and desensitized ovary. Immunolocalization at the electron microscopic level showed that SR-BI was associated with microvilli and microvillar channels of the luteal cell surface. This result supports the hypothesis that microvilli and microvillar channels represent a cell surface compartment that is specialized for the selective uptake of lipoprotein cholesterol into steroidogenic cells.

Animals↗

PubMed2003

Ephrin B1 is expressed on human luteinizing granulosa cells in corpora lutea of the early luteal phase: the possible involvement of the B class Eph-ephrin system during corpus luteum formation.

Ephrins and their Eph receptors are both membrane-bound proteins that function in various cell-cell recognition processes, such as morphogenesis and angiogenesis. In this study we examined the expression of B class ephrins-Ephs in the human ovary during corpus luteum formation, a process of tissue remodeling accompanied by angiogenesis. RT-PCR analysis detected mRNAs for Eph B1, B2, and B4 and ephrin B1 and B2, but not Eph B3 and B6 or ephrin B3, in human corpora lutea of the early luteal phase. By immunohistochemistry, ephrin B1 was moderately expressed on theca interna cells, but was expressed at a low level on granulosa cells in the preovulatory follicles. After ovulation, a rapid increase in ephrin B1 expression was observed on luteinizing granulosa cells, whereas its expression on luteinizing theca interna cells decreased. The mRNA expression of ephrin B1 in luteinizing granulosa cells was confirmed by Northern blotting. Flow cytometry showed that ephrin B1 was expressed on the surface of isolated luteinizing granulosa cells. Moreover, these cells had the ability to bind to recombinant Eph B2-Fc fusion protein. These findings suggest that ephrin B1-expressing granulosa cells can directly interact with Eph-bearing cells during corpus luteum formation in vivo, suggesting that Eph-ephrin system is involved in this process.

Adult↗

PubMed2004

Epidermal growth factor receptor inhibition by tyrphostin 51 induces apoptosis in luteinized granulosa cells.

Growth factors may be involved in the control of ovarian cell fate and could contribute to regulation of ovarian cell apoptosis. Our objective is to test the hypothesis that, in human luteinized granulosa cells, epidermal growth factor (EGF) works through the MAPK signaling pathway and inhibition of EGF receptor by a specific tyrosine kinase inhibitor, tyrphostin 51, will inhibit the activation of MAPK and induce apoptosis. Luteinized granulosa cells from human in vitro fertilization aspirates were treated as follows: 1) vehicle (dimethylsulfoxide:ethanol), 2) EGF, 3) tyrphostin 51, and 4) tyrphostin 51 plus EGF. Blockage of EGF receptor by tyrphostin 51 reduced the MAPK activity and inhibited phosphorylation and nuclear translocation of activated MAPK. Blockage of EGF receptor also induced apoptosis as demonstrated by the activation of caspase-3, an executioner protease of the apoptotic pathway, and by an increased percentage of subdiploid apoptotic nuclei. These results support the hypothesis that in human luteinized granulosa cells, EGF works through the MAPK signaling pathway and that its inhibition by tyrphostin 51 inhibits MAPK phosphorylation and induces apoptotic nuclear changes. Our data thus provide additional information regarding regulation of apoptosis in luteinized granulosa cells.

Apoptosis↗

PubMed1993

Concerted action of human chorionic gonadotropin and norepinephrine on intracellular-free calcium in human granulosa-lutein cells: evidence for the presence of a functional alpha-adrenergic receptor.

Luteal cells are known to possess receptors for LH/hCG and receptors of the beta-adrenergic type. Interactions of specific agonists with either receptor lead to the activation of adenylate cyclase and subsequently to an increase of cAMP. Since in the human there is also evidence for the presence of alpha-adrenergic receptors, we have investigated whether activation of these receptors is linked to calcium as a second messenger and performed measurement of intracellular free calcium (Ca2+) with Fura-2 in single human granulosa-lutein cells. Addition of either hCG (100, 1,000, 25,000 IU/L) or norepinephrine (NE; known to interact with both alpha- and beta-adrenergic receptors), beta-adrenergic receptor agonist isoproterenol (ISO), or alpha-adrenergic receptor agonist phenylephrine (PHE; all at 10 and 100 mumol/L) did not increase free intracellular Ca2+. However, the addition of combinations of NE/hCG, PHE/hCG, but not the combination ISO/hCG, induced a transient increase in cytosolic free Ca2+. The NE/hCG-evoked calcium signal was not abolished in the presence of the beta-adrenergic receptor antagonist propranolol and was not affected by removal of extracellular Ca2+. Furthermore, we tested whether catecholamines affected the release of progesterone in the presence or absence of hCG. As expected, hCG (10,000 IU/L) stimulated progesterone release by cultured granulosa-lutein cells. When these cells were incubated with NE, PHE, or ISO (at 10 mumol/L), production of progesterone by these cells was not affected. However, the combinations of NE and PHE with hCG abolished the hCG-induced progesterone accumulation, but ISO coincubated with hCG did not. Taken together, our results indicate: 1) the presence of functional alpha-adrenergic receptors on human granulosa-lutein cells; 2) simultaneous activation of two different receptors (for hCG and alpha-agonists) are able to evoke intracellular Ca2+ elevation, implicating postreceptor interactions in human granulosa lutein cells; 3) this process occurs even in the absence of extracellular Ca2+, indicating the involvement of intracellular Ca2+ stores, most likely due to activation of phosphoinositide pathway; 4) catecholamines most likely acting via alpha-adrenergic receptors, inhibit the LH/hCG-induced release of progesterone.

Calcium↗

PubMed1993

Effect of oxytocin on free intracellular Ca2+ levels and progesterone release by human granulosa-lutein cells.

Oxytocin and its receptor are found in the corpus luteum in a variety of species, including the human. In the present study we used fura-2 microfluorimetry to investigate whether activation of the oxytocin receptor of cultured human granulosa-lutein cells causes intracellular calcium (Ca2+) signals and affects progesterone release. Although after 1 day in culture, cells were not responsive to oxytocin, the number of responsive cells increased steadily during the first 3 days in culture, reaching a maximum on days 4 and 5 (59-66%) and then declined again until day 8. Effective oxytocin concentrations were apparently independent of the culture day, and concentrations as low as 10 nmol/L increased intracellular free Ca2+ levels from 70-140 nmol/L (basal levels) to maximal peak levels of 800 nmol/L. The oxytocin-induced Ca2+ signal was not affected by removal of extracellular Ca2+ with EGTA. Moreover, depletion of intracellular Ca2+ stores by ionomycin treatment rendered the cells unresponsive to oxytocin, pointing also at the intracellular source of the oxytocin-inducible Ca2+ signal. Interestingly, after one single stimulation with oxytocin, cells became refractory to additional stimuli, and only extremely high concentrations of oxytocin induced a second increase in intracellular free Ca2+. To examine the possible effects of oxytocin on progesterone release by cultured cells, we incubated cells on culture day 2 (20% responsive cells in the fura measurements) and culture day 5 (66% responsive cells in the fura measurements) for 24 h with oxytocin (10 nmol/L) and hCG (10,000 IU/L). Although hCG significantly stimulated progesterone release, oxytocin alone was without a stimulatory effect on either day. However, a significant augmentation of the effect of hCG on progesterone release was found in incubations of cells on day 5. Interestingly, the effects of hCG also included stimulation of oxytocin release by cultured granulosa-lutein cells into the culture medium, as determined by RIA. In summary, our data indicate the presence of a functional oxytocin receptor on human granulosa-lutein cells that is linked to Ca2+ as a second messenger released from intracellular Ca2+ stores. The number of oxytocin-responsive cells increases during differentiation in culture. Moreover, oxytocin release induced by hCG and a stimulatory effect of oxytocin on the hCG-induced progesterone production during the period of maximal responsiveness of cultured cells were found. We, therefore, propose that oxytocin may have autocrine and/or paracrine functions in human granulosa-lutein cells, including fine-tuning of progesterone release.

Calcium↗