Cultured Granulosa-lutein Cells Research Articles

Expression of adrenomedullin by human granulosa lutein cells and its effect on progesterone production.

Adrenomedullin (AM) has diverse functions and is expressed in a variety of tissues. This study was conducted to investigate the expression of AM in the human ovary and its effect on progesterone production by human granulosa lutein cells. Follicular fluid and blood samples were obtained at the time of oocyte retrieval from patients undergoing in vitro-fertilization cycles. Concentrations of AM in follicular fluid and plasma were measured by RIA. Granulosa cells were isolated from follicular fluid and expression of AM mRNA was examined by RT-PCR. Granulosa lutein cells were cultured in vitro and secretion of AM by those cells was determined by immunoprecipitation followed by PAGE. Immunohistochemical staining with human ovaries was carried out, using a specific antibody to AM. Furthermore, the effect of AM on progesterone production by cultured granulosa lutein cells was studied. Concentrations of AM in follicular fluid collected just before ovulation were significantly higher than those in the plasma (P<0.01). AM mRNA was expressed in granulosa cells at the preovulatory stage. Cultured granulosa lutein cells secreted immunoreactive AM. With immunohistochemical staining, it was revealed that AM was most abundantly expressed in granulosa lutein cells at the midluteal phase. No appreciable staining for AM was observed in granulosa cells in primordial and preantral follicles, whereas immunolocalization of AM was noted in granulosa cells of dominant follicles although it was not as prominent as in granulosa lutein cells at the midluteal phase. Furthermore, addition of AM to cultured granulosa lutein cells augmented progesterone secretion in a dose-dependent manner. These results suggest that AM is transcribed and secreted in human granulosa lutein cells as a local factor to enhance progesterone production by those cells.

Impaired insulin action on granulosa-lutein cells in women with polycystic ovary syndrome and insulin resistance

We studied the in vitro response to insulin of granulosalutein cells derived from patients with polycystic ovary syndrome (PCOS) and clinically defined insulin resistance. Insulin sensitivity was assessed by continuous infusion of glucose with model assessment test (CIGMA). Insulin resistant (PCOS-IR; n = 8), non-insulin resistant (PCOS-NIR; n = 9) patients with PCOS, and women with tubal factor infertility (TF; n = 8) underwent controlled ovarian stimulation with long-term gonadotropin-releasing hormone (GnRH) agonist, recombinant follicle stimulating hormone (FSH), and in vitro fertilization. Primary cultures of granulosa-lutein cells were incubated with insulin (10, 100, 500 ng/ml) and/or luteinzing hormone (LH) (10, 100 ng/ml) in the presence of low density lipoprotein (100 μg/ml). The progesterone and lactate accumulation were measured in the culture medium. LH potently stimulated the progesterone secretion in all groups. Insulin alone had no effect on progesterone release in any of the groups, but stimulated lactate formation in the PCOS-NIR and TF groups. Insulin augmented the effect of LH on progesterone secretion selectively in the PCOS-NIR group. The expression of the insulin receptor was determined by Western blotting in separate cultures of granulosa-lutein cells, and showed receptor down-regulation in the PCOS-IR patients. We infer that the in vitro effect of insulin on progesterone and lactate release by granulosa-lutein cells is impaired in insulin resistant PCOS patients.

Detection of toxins to human female reproduction using human luteinized-granulosa cells in culture

Discharge of man-made organic compounds into the environment poses unknown risks to human reproduction. With less than 10% of the estimated 80,000 man-made chemical compounds having been specifically tested for toxic reproductive effect, an accurate and routine testing procedure is critical to further evaluate the risks associated with these compounds. Previous research from the Department of Obstetrics/Gynecology, Texas Tech University and other laboratories has suggested that cultured human luteinized-granulosa cells (HLGC) may be useful as a model for toxicology studies. In the current study, HLGC were exposed to varying levels of four organic solvents with known or suspected carcinogenic potential. Using the established culture system, HLGC collected from patients in the in vitro fertilization program were collected and cultured at known densities. Once the cultures were established, the wells were rinsed and the media replaced with media containing increasing concentrations of the organic solvents acetone, benzene, carbon tetrachloride, and toluene. After 48 hours the media was removed for hormonal analysis and the cells were prepared for electron microscopy. Using a random complete block design with factorial arrangement of treatments, the resulting data were statistically analyzed using analysis of variance and Fisher's polynomial least significant difference for mean separation. There was an overall trend toward decreased progesterone production ( P = .14) with increasing compound concentration. As might be expected, these effects were compound specific ( P < .004), with increasing levels of toluene appearing to have the most deleterious effect ( P < .001). Ultrastructural examination revealed leaching of cell lipids by all compounds at concentrations of 10 ppm and higher. This decreased production of progesterone would affect embryo implantation and uterine viability, which could explain the increased incidence of spontaneous abortions in women routinely exposed to these compounds.

Leukocytes modulate 11 β-hydroxysteroid dehydrogenase (11 β-HSD) activity in human granulosa-lutein cell cultures

It is well established that there are interactions between the immune and reproductive systems. The ovary contains indigenous macrophages, as well as other classes of leukocytes in smaller numbers. Cytokines secreted by these cells have been shown to have the ability to regulate ovarian steroidogenesis. In the present study, the effect of leukocytes on 11 β-hydroxysteroid dehydrogenase (11 β-HSD) in human granulosa-lutein cells was examined. In addition, individual cytokines were also tested for their ability to regulate this enzyme. The follicular aspirates of patients undergoing IVF treatment were used as a source of granulosa cells. Cells isolated from these aspirates were found to contain between 15 and 60% leukocytes as assessed by flow cytometry (FACS). Leukocytes were removed from the sample preparations by the use of immunomagnetic beads coated with CD45 antibody, which recognises a surface antigen on all classes of leukocyte. Removal of leukocytes significantly decreased the 11 β-HSD activity in the granulosa cells, assayed after 3 days of culture, from 7.3 (2–20) to 3.5 (1–10) pmol cortisone formed/50 000 cells/4 h (medians and ranges, n=15). Addition of IL-5 and IL-6 significantly increased the 11 β-HSD activity in granulosa cell cultures both in the presence and absence of leukocytes. Addition of IL-4 and IFN- γ increased ll β-HSD activity only in the leukocyte-depleted granulosa cell cultures, whereas IL-2 had no effect on either of the cultures. The data suggests that leukocytes interact with the ovarian cells through cytokine secretion and/or cell–cell contact to increase the 11 β-HSD activity in human granulosa cells.

Simulation of human luteal endocrine function with granulosa lutein cell culture.

Human granulosa cells collected from in vitro fertilization have previously been cultured to provide a system to simulate the granulosa lutein cells of the corpus luteum. In most of these systems, the cultures have been relatively short term, and attempts to simulate the normal pattern of hormone production observed during the luteal phase of the cycle have not been reported. Additionally, the hormone relaxin has generally been absent from the endocrine analysis of these systems. In this report, methods were used that supported secretion of ovarian steroids and relaxin that mimics the profiles of these hormones in vivo. This system was used to observe the endocrine responses of the granulosa lutein cells to three different protocols of CG administration designed to mimic the normal luteal phase, early pregnancy, and early pregnancy followed by pregnancy loss. The normal luteal phase was simulated by a constant baseline (0.02 IU/mL) CG model to simulate a nonconceptive cycle (baseline). The second model was baseline CG until day 8 of culture, followed by daily doubling from days 9-17 to simulate an early pregnancy (rescue-plateau). CG concentrations were then held constant from days 17-20 (5.12 IU/mL). A third model (rescue-drop) was used that was identical to the early pregnancy model except that on day 17 CG was returned to baseline concentrations (0.02 IU/mL) to simulate an early pregnancy loss. Baseline CG stimulation resulted in profiles of estrogen, progesterone, and relaxin secretion in culture that were closely related to secretory profiles previously reported in serum during the nonconceptive luteal phase. The timing of appearance of relaxin secretion and later declines in steroid and relaxin secretion paralleled that observed in serum. In the CG rescue protocols, ovarian steroids rose in response to daily doubling of CG and fell when CG either plateaued or fell. Relaxin did not show an increase in response to increasing CG, but its secretion did not drop when CG concentrations plateaued or dropped. This cell culture system model mimics the profile of ovarian steroids and relaxin seen in serum during the nonconceptive luteal phase, although the relative magnitude of the hormones was not the same as seen in vivo. It was also used to investigate responses to luteal rescue protocols designed to simulate early pregnancy and pregnancy loss. This culture system may be useful to study differences in endocrine response in granulosa cells collected from different patients and to provide information of clinical relevance. This culture system provides a model to study luteal function and its response to different protocols of luteal rescue and thus may provide insight into early pregnancy and pregnancy loss.

Simulation of Human Luteal Endocrine Function with Granulosa Lutein Cell Culture

Simulation of Human Luteal Endocrine Function with Granulosa Lutein Cell Culture

Monocyte chemotactic protein-1 expression in human preovulatory follicles and ovarian cells

There is a considerable population of macrophages (5–15% of the cells) within the human ovarian follicle at the time of ovulation. Macrophages are also present within the ovarian stroma, mostly near perifollicular capillaries. We hypothesized that macrophage migration in and around the preovulatory follicle is hormonally regulated and that regulation of macrophage migration occurs through local modulation of monocyte chemotactic protein-1 (MCP-1) that chemoatracts and activates monocytes/macrophages. In this regard, we investigated the expression and regulation of MCP-1 in human follicular fluid and in ovarian stromal and granulosa-lutein cell cultures. The concentration of MCP-1 in follicular fluid samples obtained from women prior to the administration of hCG was ( n=4) 90±27 (mean±S.E.) pg/ml; in samples obtained 12 h after the hCG administration it was ( n=3) 135±23 pg/mL; in follicular fluids obtained 34 h after the hCG administration it was ( n=126) 322±46 pg/mL ( P=0.007 vs. pre-hCG). The mean ratio of follicular fluid/serum MCP-1 levels was 4.18. There was a correlation between follicular fluid MCP-1 levels and follicular fluid or serum progesterone levels ( r=0.21, P=0.02; r=0.29, P=0.03, respectively). MCP-1 mRNA and the protein were expressed in ovarian stromal and granulosalutein cells in culture and were increased by interleukin-1α and tumor necrosis factor-α in a time- and concentration-dependent manner. LH hCG induced higher levels of MCP-1 mRNA expression and protein production in both cell cultures. We propose that regulation of MCP-1 in ovarian stromal and granulosa-lutein cells by cytokines may play a role in the physiology of periovulatory events.

Interleukin-8 expression and modulation in human preovulatory follicles and ovarian cells.

Just before the time of ovulation, the number of neutrophils increases markedly in the thecal layer of the leading follicle. A preovulatory rise in chemotactic activity for neutrophils in human follicular fluid has also been detected. We hypothesized that interleukin-8 (IL-8), a neutrophil chemoattractant/activating factor and a potent angiogenic agent, may be an important modulator of leukocyte chemotaxis in ovulatory function. In this regard we investigated the expression and modulation of IL-8 in human follicular fluid samples from patients undergoing in vitro fertilization-embryo transfer therapy and in ovarian stromal and granulosa-lutein cell cultures. The concentration of IL-8 in pre-hCG follicular fluid samples (n = 4) was 16 +/- 12 (mean +/- SEM) pg/ml, and that in post-hCG samples (n = 101) was 262 +/- 45 pg/ml (P = 0.001). In post-hCG samples, the concentration of IL-8 in an individual follicle correlated with the size of that follicle (r = 0.61; P = 0.02). We also observed a correlation between serum IL-8 levels (22 +/- 3 pg/ml) and follicular fluid levels (303 +/- 143 pg/ml), with a 14-fold gradient (r = 0.71; P = 0.01) in 11 patients tested for both. IL-8 messenger RNA (mRNA) and the protein were expressed constitutively in ovarian stromal cell cultures, and the level was increased by IL-1 alpha and tumor necrosis factor-alpha in a time- and concentration-dependent manner. hCG and LH induced higher levels of IL-8 mRNA expression and protein production. Granulosalutein cells also expressed IL-8 mRNA and protein, and the levels were increased by IL-1 alpha and tumor necrosis factor-alpha. Importantly, progesterone suppressed both basal and IL-1 alpha-stimulated IL-8 expression in stromal and granulosa-lutein cell types. In summary, we found that IL-8 levels are elevated in periovulatory follicular fluid, and both granulosa-lutein and ovarian stromal cells express the mRNA and produce the protein. The modulation of IL-8 in these cell cultures by steroid and trophic hormones suggests that IL-8 may play an important role in the physiology of ovulation, such as timely follicular rupture and neovascularization of the corpus luteum.

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.

Granulocyte-macrophage colony-stimulating factor: presence in human follicular fluid, protein secretion and mRNA expression by ovarian cells.

In recent years it has become evident that a leukocyte-cytokine network contributes to the paracrine regulation of ovarian function. The objectives of this study were to examine the presence of a potent lympho-haemopoietic cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), in tissues and fluids from human ovaries. In a prospective study, follicular fluid and plasma were collected from naturally cycling women and women undergoing hyperstimulation for in-vitro fertilization (IVF). Granulosa-lutein cells were collected at the time of oocyte recovery for IVF and corpora lutea were collected at the time of hysterectomy for non-ovarian reasons. Culture supernatants from ovarian cell and tissue cultures were harvested on completion of a 48 h incubation. Immunoactive GM-CSF was measured by enzyme-linked immunosorbent assay, and was found to be present at statistically significantly higher levels in follicular fluid (8.9 +/- 0.7 pg/ml) and plasma (11.3 +/- 0.8 pg/ml) of women undergoing hyperstimulation compared to follicular fluid (5.3 +/- 0.3 pg/ml) and plasma (7.1 +/- 0.5 pg/ml) from naturally cycling women. Immunoactive GM-CSF was also detected in culture supernatants of granulosa-lutein cells (47.6 pg/10(5) cells), early luteal phase corpora lutea (0.52 pg/microgram DNA) and mid-luteal phase corpora lutea (0.98 pg/microgram DNA). Furthermore, transcripts for GM-CSF, and both the alpha and beta subunits of the GM-CSF receptor, were detected by reverse transcription polymerase chain reaction (RT-PCR) in granulosa-lutein cell culture preparations and corpora lutea collected during the early, mid- and late luteal phase of the menstrual cycle. These results show that GM-CSF is expressed and secreted by cells within the human ovary, and, together with the finding of expression of mRNA for GM-CSF receptor, suggest a role for GM-CSF in the local regulation of ovarian events.

Interleukin-1-mediated stimulation of prostaglandin E production is without effect on plasminogen activator activity in human granulosa lutein cell cultures.

In continuation of earlier observations on the involvement of interleukin-1 (IL-1) in ovarian function, we examined the ability of IL-1 to modulate plasminogen activator (PA) activity and prostaglandin (PG) synthesis in human granulosa lutein cells (GLCs). Toward this goal, GLCs were obtained from women undergoing in vitro fertilization, preincubated with 10% fetal calf serum for 48 h, and subsequently cultured for 48 h in serum-free media in the absence or presence of IL-1 beta (10 ng/mL). Cellular PA activity was measured by plasminogen-dependent cleavage of the chromogenic substrate H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251). Prostaglandin E (PGE) levels were assayed by conventional RIA. Exposure of GLCs to IL-1 resulted in a 50% increase in PGE production, a 33% suppression of PA activity, and a 75% increase in the ability of the corresponding conditioned media to inhibit exogenous urokinase activity. The inhibitory capacity was attributable to an IL-1-mediated increase in PA inhibitor type-1 (PAI-1) production, inasmuch as urokinase inhibition could be abolished by the administration of a polyclonal antihuman PAI-1 immunoglobulin G. IL-1 treatment had no effect on plasmin or trypsin inhibition. Exposure of GLCs to IL-1 receptor antagonist abolished the ability of IL-1 to enhance PA inhibitory activity and PGE production, thereby establishing specific IL-1 receptor-mediated effects. The ability of IL-1 to suppress PA activity and to produce PAI-1 persisted in the presence of indomethacin, a potent inhibitor of PG synthesis. Likewise, transforming growth factor-beta 1 suppressed the ability of IL-1 to stimulate PGE production without affecting the IL-1-induced effects on the PA system. The present findings suggest a pluripotent response of GLCs to IL-1, characterized by the induction of PAI-1 and the suppression of PA occurring concurrent with, but independent of, PG production. These observations support the potential involvement of IL-1 in the regulation of human ovulatory processes.

Interleukin-1-mediated stimulation of prostaglandin E production is without effect on plasminogen activator activity in human granulosa lutein cell cultures

Interleukin-1-mediated stimulation of prostaglandin E production is without effect on plasminogen activator activity in human granulosa lutein cell cultures

Regulation of 72-kd type IV collagenase-matrix metalloproteinase-2 by estradiol and gonadotropin-releasing hormone agonist in human granulosa-lutein cells

To examine the hormonal regulation of 72-kd type IV collagenase-gelatinase-matrix metalloproteinase-2 in human granulosa-lutein cells. The effect of E2 and GnRH agonist (GnRH-a) on matrix metalloproteinase-2 amount was examined on a long-term granulosa-lutein cell cultures. The preovulatory follicles from women participating in our IVF program were aspirated through the vaginal wall. Follicular fluids were centrifuged and the granulosa cells were separated and pooled for culturing. Matrix metalloproteinase-2 enzyme activity was assayed with type IV collagen degradation assay and zymography. The matrix metalloproteinase-2 immunoreactive protein was studied by using specific antibodies. Matrix metalloproteinase-2 was the predominant form of gelatinase in cultured granulosa-lutein cells. The enzyme activity as well as the immunoreactive protein increased in the culture medium of the granulosa-lutein cells in the presence of E2. The effect of E2 was abolished by GnRH-a. Estradiol regulates the matrix metalloproteinase-2 enzyme amount in granulosa-lutein cells; the effect may be antagonized by GnRH-a.

Interleukin-1-mediated suppression of plasminogen activator activity in human granulosa lutein cell cultures is not mediated by prostaglandin e production

Interleukin-1-mediated suppression of plasminogen activator activity in human granulosa lutein cell cultures is not mediated by prostaglandin e production

Estradiol and progesterone production by cultured granulosa cells cryopreserved from in vitro fertilization patients.

Gonadotropin-stimulated steroidogenesis was studied in cultured human granulosa-lutein cells obtained from patients undergoing procedures for in vitro fertilization. The impact of cryopreservation on cell function in vitro was studied. Granulosa cells obtained from in vitro fertilization patients were cultured in serum-supplemented medium or cryopreserved at -135 degrees C for 2-22 months. Fresh (unfrozen) cells (10(5) produced estradiol at a rate of 1320 pmol/l (over 72 h) and progesterone at about 2500 nmol/l. Estradiol production by either fresh or cryopreserved granulosa cells in culture was unaffected by physiological concentrations of follicle-stimulating hormone (7 IU/l). Adding testosterone (10(-7) mol/l) to the medium increased estradiol secretion approximately sixfold. In contrast, progesterone production was not affected by follicle-stimulating hormone or testosterone. No significant differences were observed in cultures of cryopreserved granulosa cells compared to cultures of unfrozen cells with respect to estradiol secretion, the effects of follicle-stimulating hormone or testosterone on estradiol secretion, or progesterone production. Progesterone production by fresh and cryopreserved cells was stimulated by human chorionic gonadotropin. These data indicate that cryopreservation offers the potential to facilitate prospective studies utilizing large numbers of human granulosa-lutein cells in culture.

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.

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.

The effect of clomiphene citrate isomers on human granulosa-lutein cells in culture.

The effect of racemic clomiphene citrate and its two individual isomeric forms (i.e. en and zu) on corpus luteum function was evaluated. Granulosa-lutein cells were obtained from three normal ovulatory women undergoing oocyte retrieval following ovulation induction with agents other than clomiphene citrate for in vitro fertilization--embryo transfer (IVF-ET). The granulosa cells were cultured in the presence and absence of the three forms of clomiphene citrate, and in the presence and absence of human chorionic gonadotropin (hCG). Patients were recruited from the unit for assisted reproductive technology in a university hospital. The main outcome measured was the production of estradiol and progesterone by cultured human granulosa cells under the various conditions described above. The production of estradiol and progesterone by the cultured granulosa cells was dose-dependently reduced to a similar extent by all three forms of clomiphene citrate. The addition of hCG augmented steroidogenesis in all groups at all concentrations, but this still remained lower in all clomiphene citrate-treated groups compared to controls. The data suggest that all three types of clomiphene citrate (racemic, en, and zu) have inhibitory effects on the production of estradiol and progesterone by cultured human granulosa-lutein cells.

Concerted action of human chorionic gonadotropin and norepinephrine on intracellular-free calcium in human granulosa-lutein cells

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.

Comparison of Functional Response of Rat, Macaque, and Human Ovarian Cells in Hormonally Defined Medium1

A serum-free medium has been developed which supports in vitro function by ovarian cells derived from rat, monkey, and human tissue. This granulosa cell medium (GCM) consists of Dulbecco's Modified Eagle's Medium: Ham's F-12 medium (1:1, v:v) supplemented with insulin, transferrin, aprotinin, selenium, fibronectin, penicillin, and streptomycin. Ovarian cells from three species were compared: rat, macaque, and human. Four types of ovarian cultures were examined: 1) purified granulosa cell cultures and 2) co-cultures containing granulosa-theca-stroma cells, 3) luteal cells, and 4) granulosa-lutein (harvested from in vitro fertilization cultures) cells. Each cell type was characterized by its response to FSH or hCG when cultured in GCM. Morphologic responses to FSH were observed in GCM in rat granulosa and granulosa-theca-stroma cell cultures, macaque and human granulosa-lutein cells, and human granulosa-theca-stroma cell cultures. The FSH-stimulated cells retracted and became rounded, leaving long intercellular connections. Luteal cells did not retract in response to FSH, and the cells remained firmly attached to the fibronectin matrix. Steroidogenic regulation of the GCM-cultured ovarian cells was monitored following stimulation of the cultures with FSH. The ability of the cells to aromatize testosterone was first examined. Rat granulosa cell cultures and granulosa-theca-stroma cell cultures, macaque granulosa-lutein cell cultures, and human granulosa-theca-stroma cell cultures all accumulated estradiol when given FSH and testosterone for 48 h. Moreover, these cell types as well as human luteal cells were able to metabolize 25-hydroxy [1,2-3H]cholesterol to various steroid metabolites. The data indicate that GCM supports normal granulosa cell morphologic response to FSH.(ABSTRACT TRUNCATED AT 250 WORDS)