Gonadotropin releasing hormone (GnRH) stimulates both secretion and synthesis of human chorionic gonadotropin (hCG) by first trimester human placental minces in vitro

An in vitro system using the minces of placental villi from first trimester human pregnancy (6-10 weeks) has been validated to examine the effect of addition of GnRH and its analogues on hCG secreted into the medium. Addition of low concentration of GnRH or its analogues (1 X 10(-8) M to 1 X 10(-6) M) resulted in an increase in the quantity of hCG in the medium, while addition of high concentrations of GnRH resulted in an inhibitory effect. Of the analogues tested, Buserelin was highly effective in exerting an inhibitory effect. A significant increase in 35S-methionine incorporation into immunoprecipitable hCG was noticed in the presence of GnRH. These results suggests that GnRH stimulates both synthesis and secretion of hCG by first trimester human placenta.     

Upregulation of placental indoleamine 2,3-dioxygenase by human chorionic gonadotropin

We tested the hypothesis that hCG can upregulate human trophoblast indoleamine 2, 3-dioxygenase (INDO), which catalyzes the breakdown of tryptophan in villous circulation. The results revealed that it can. Treatment of human trophoblasts with hCG resulted in a time and dose dependent increase in INDO mRNA and protein levels and its enzyme activity. The hCG effect was hormone specific and required the dimer conformation of hCG. The hCG effect required its receptors and was mediated by a cAMP dependent, but protein kinase A independent, mitogen-activated protein kinase 3/1 (MAPK3/1) signaling mechanism. In summary, the present data demonstrate a novel hCG effect on human placental INDO, which probably plays a key role at maternal fetal interface in preventing fetal rejection.     

Effect of FSH and HCG on progesterone secretion by cultured oocyte-cumulus complexes and granulosa cells from porcine antral follicles: Influence of follicular development

Oocyte-cumulus complexes and granulosa cells were harvested from small (1–2 mm), medium (3–5 mm), and large (6–12 mm) porcine antral follicles and cultured for 2 and 3 days. The effects of various doses of purified hCG and human FSH on progesterone secretion and monolayer formation were examined. After a 2-day culture period it was found that FSH was more effective in stimulation of progesterone secretion by cultured oocyte-cumulus complexes than in granulosa cells harvested from small follicles (P < 0.01), whereas hCG was more effective in stimulating progesterone secretion in granulosa cells than in oocytecumulus complexes harvested from large follicles. In contrast, after a 3-day culture period, granulosa cells secreted more progesterone compared to oocytecumulus complexes under control conditions or in the presence of hCG or FSH. After 3 days both FSH and hCG stimulated progesterone secretion by oocytecumulus complexes and granulosa cells; however, the hormone effect was greater upon granulosa cells than oocyte-cumulus complexes. After 3 days of culture in the case of both follicular cell types, there was a greater response to FSH in the case of cells harvested from small compared to large follicles. The reverse was true in the case of hCG responsiveness. Monolayer formation ability of oocyte-cumulus complexes was greater in the case of complexes harvested from small and medium than complexes harvested from large follicles. Addition of hCG to the cultures led to a dose-dependent decrease in monolayer formation by oocyte-cumulus complexes harvested from all sizes of follicles.     

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.     

Evidence for a Sertoli cell, FSH-suppressible inhibiting factor(s) of testicular steroidogenic activity

By using a model of immature porcine Leydig and Sertoli cells cultured in serum free defined medium, we evidenced a paracrine control of Leydig cell steroidogenic activity by Sertoli cells via a secreted inhibiting protein(s). This protein(s), partially purified using gel filtration (M.W. 20,000-30,000) suppresses the steroidogenic responsiveness to LH/hCG by decreasing the specific LH/hCG binding (52% decrease) and hormone steroid biosynthesis (73% decrease) at a level(s) located between cAMP production and pregnenolone formation. The suppression of this inhibitor(s) by FSH, in a dose dependent manner, is one mechanism by which FSH "sensitizes" Leydig cell response to LH/hCG stimulation.     

Development and characterization of an in vitro ovulation model using mouse ovarian follicles.

To investigate ovulation, an in vitro model with cultured mouse follicles was developed and compared with an in vivo ovulation model. In this model, secondary follicles were grown in vitro with immature mouse serum (5%) and recombinant human FSH. Addition of ascorbic acid and selenium to the medium increased follicular survival (from 29% to 86%) and resulted in the development of healthy preovulatory follicles (> 400 microm) producing estradiol. Depending on the starting size of the follicles, the preovulatory stage was reached after 4-6 days. The ovulatory response to hCG was maximal in follicles exceeding a diameter of 400 microm. The in vitro-ovulated oocytes could be fertilized and were able to develop to the blastocyst stage. Ovulation induced by hCG was dose dependent, reaching a maximum of 80% at 1 IU/ml. Concomitantly, progesterone production increased from 3.6 +/- 0.5 to 29 +/- 2 ng/ml. Both in vivo and in vitro, hCG induced expression of the progesterone receptor and the prostaglandin endoperoxide synthase-2 (PGS-2) gene within 3 h. Ovulation could be completely blocked with the anti-progestogen Org-31710 and partially (50%) with the PGS inhibitor indomethacin in vitro and in vivo. Org-31710 and indomethacin did not affect progesterone production. In summary, a physiologically relevant in vitro ovulation model of cultured mouse follicles that can be used to study the process of follicular rupture has been developed.     

Specific low density lipoprotein receptors in pig Leydig cells. Role of this lipoprotein in cultured Leydig cells steroidogenesis

Freshly prepared and cultured pig Leydig cells were shown to possess specific binding sites for iodinated human low density lipoprotein (LDL). Binding of LDL was followed by internalisation. Both processes were inhibited by unlabelled LDL but not high density lipoprotein (HDL). The number of LDL binding sites was enhanced by prior treatment of cultured cells with hCG. Addition of LDL to the culture medium caused a large enhancement of the rate of steroid secretion (testosterone and dehydroepiandrosterone sulfate) both in the presence or absence of hCG. On the contrary, HDL decreased the steroid output, both in the presence or absence of hCG. We concluded that LDL cholesterol rather than cholesterol synthesized $\text{de novo}$ by the cells, is the major substrate for androgen production by pig Leydig cells in culture.     

Effect of calcium on corticosteroid secretion by isolated frog interrenal gland

The direct effect of extracellular calcium concentrations on corticosteroidogenesis has been examined in the frog, using a perifusion system technique. The release of corticosterone and aldosterone in the effluent medium was monitored by specific radioimmunoassays. Increasing concentrations of Ca2+ (from 2 to 15 mM) gave rise to a dose-related stimulation of corticosteroid release, whereas the increment of either Na+ or K+ concentrations did not modify steroid production. Iterative administration of a moderate concentration of calcium (6 mM) led to a reproducible stimulation of steroid secretion whereas the same dose infused during 6 h induced a transient rise in corticosteroid secretion followed by a plateau. The direct effect of Ca2+ on steroidogenesis was confirmed by the dose-dependent stimulation of steroid secretion induced by the calcium ionophore A 23187. Perifusion with a calcium-free medium or blockade of Ca2+ channels by 4 mM Co2+ both resulted in a significant decrease in steroid production. Conversely, the administration of verapamil (up to 10(-4) M) did not affect steroidogenesis. These results provide evidence that extracellular calcium ions are required for basal production of corticosteroids in amphibians and that Ca2+ influx does not occur through voltage-dependent channels. Since, in the frog, blood Ca2+ concentrations vary in a rather large range, these results suggest that circulating Ca2+ levels may regulate corticosteroid production in these animals.     

Ovarian steroidogenic responsiveness to exogenous gonadotropin stimulation in young and middle-aged female rats

Reproductive aging in the female rat is associated with gradual declines in LH secretion and ovarian progesterone (P) production. This study examined whether the influences of aging on P levels reflect decreased ovarian responsiveness to gonadotropin stimulation, as opposed to changes in gonadotropin release. Young and middle-aged regularly cyclic female rats received sodium pentobarbital to block endogenous proestrous luteinizing hormone (LH) surges, followed by administration of various doses of human chorionic gonadotropin (hCG). Similar treatments were performed in middle-aged acyclic persistent-estrous (PE) females. Injection of hCG resulted in equivalent plasma hCG levels in each treatment group. At the lowest hCG dose tested, a significant rise in plasma P levels was observed in middle-aged cyclic rats, but not in young cyclic or middle-aged PE females. This unexpected finding may reflect accelerated follicular development in middle-aged cyclic females, as suggested by a previous study. At the intermediate dose, young and middle-aged cyclic but not PE rats displayed significantly increased P in response to hCG. At the highest dose tested, all three groups of rats displayed increased P levels after hCG stimulation. However, P concentrations were significantly lower in middle-aged PE than regularly cyclic females. Northern and slot blot hybridization analyses revealed that ovarian mRNA levels for cytochrome P450 side-chain cleavage, the rate-limiting enzyme in P synthesis, were markedly reduced in PE rats following hCG stimulation. These findings indicate that ovarian responsiveness to gonadotropin stimulation is impaired in middle-aged PE, but not regularly cyclic rats, and suggest influences of cycle status on the biochemical and molecular mechanisms regulating ovarian steroid production. Furthermore, these findings reveal that attenuated P production in middle-aged proestrous rats is due to attenuated preovulatory LH surges, rather than decreased ovarian sensitivity to LH.     

Regulation of progesterone secretion in human syncytiotrophoblast in culture by human chorionic gonadotropin.

In the present investigation we sought to define the specific sites in the pathway of placental progesterone biosynthesis that underlie the action of human chorionic gonadotropin (hCG). When the cells were challenged with dibutryl cAMP (dbcAMP), forskolin or isobutylmethylxanthine, they produced significantly higher amounts of progesterone which in the presence of the hCG antibody was reduced to the level of the control set of cells. Trophoblast cells cultured in serum free medium with 25-hydroxycholesterol (25-OHC) produced increased amounts of progesterone. In the presence of hCG antibody at a concentration which neutralized the secreted hCG, the steroid production was completely blocked, even when the 25-OHC was added to the medium. Also, direct quantitation of the cytochrome P450 SCC enzyme in the absence of hCG indicated a significant decrease. The exogenous addition of low density lipoproteins (LDL) increased the progesterone secretion by the trophoblast cells in culture. Neutralization of hCG by the antibodies, however, drastically reduced the LDL induced progesterone secretion, which was restored by the addition of dbcAMP to the medium. Based on these findings, we suggest a stimulatory effect of hCG on normal trophoblast cells at the level of LDL utilization and cytochrome P450 SCC enzyme. Since dbcAMP could mimic these actions of hCG, the data suggest a possible autocrine/paracrine role of hCG on the trophoblast cells. An additive effect of hCG and cAMP on progesterone secretion observed in our studies, indicate that apart from hCG, adenylate cyclase activity may also be regulated by other factors.     

Modulation of secretion of human chorionic gonadotropin by biologic response modifiers on term placenta and choriocarcinoma cells

The effects of biologic response modifiers such as interferon-gamma, tumor necrosis factor alpha (TNF), and retinoic acid on the human chorionic gonadotropin (hCG) secretion of cultured choriocarcinoma cells (JAR) and term placenta have been studied. Although the proliferation of JAR cells was not inhibited by these agents, retinoic acid and TNF markedly increased both the intracellular levels as well as the secreted amounts of hCG. In the case of the term placenta, only retinoic acid increased the hCG secretion into the culture medium, whereas interferon-gamma and TNF both markedly reduced secretion. The cytostatic agent etoposide (VP-16) was able to augment the hCG secretion on the choriocarcinoma cells but did not alter its production on term placenta. The The data presented indicate different mechanisms of regulation of hCG secretion in the normal and malignant trophoblast.     

Direct demonstration of increased intracellular concentration of free calcium in rabbit and human neutrophils following stimulation by chemotactic factor

An increase in the level of intracellular free calcium concentration in rabbit and human neutrophils stimulated by chemotactic factors has been demonstrated directly using the calcium-sensitive fluorescent probe quin-2. Addition of f-Met-Leu-Phe (10(-9) M), C5a (3 x 10(-9) M) or leukotriene B4 (6 x 10(-8) M) to the neutrophils induces a rapid increase in the intracellular concentration of free calcium that reaches a maximum value 15 seconds following stimulation. At concentrations of f-Met-Leu-Phe less than 10(-8) M the enhancement is dose dependent with an ED50 of 8 x 10(-11) M and is significantly reduced in the presence of EGTA in the suspending medium.     

Regulation of apolipoprotein E synthesis in rat ovarian granulosa cells

Apoprotein E (apo-E) is a surface component of several classes of plasma lipoproteins. It functions as a ligand for receptor-mediated uptake of lipoproteins. Granulosa cells from ovaries of diethylstilbestrol-stimulated hypophysectomized immature rats cultured in serum-free medium with [35S]methionine secretes a 34-kDa protein which reacts with a monospecific anti-rat apo-E antibody and represents 0.2% of total secreted protein. Protease mapping confirms that this protein is apoprotein E. The secreted apoprotein E may be complexed with lipid since it floats in the ultracentrifuge at density less than 1.21 micrograms/ml. Freshly isolated granulosa cells contain receptors for follicle stimulating hormone (FSH) but not for human chorionic gonadotropin (hCG) or prolactin. Apoprotein E secretion is stimulated 2-fold by FSH, but hCG and prolactin have no effect. When granulosa cells develop hCG and prolactin receptors after 48 h of culture with FSH, apoprotein E secretion is not stimulated by addition of FSH, hCG, or prolactin although steroidogenesis is induced. The addition of 10(-7) M androgen plus FSH stimulates a marked increase in progestin synthesis over FSH alone, but androgen has little added effect on apoprotein E secretion. Cholera toxin (1.25 micrograms/ml) and dibutyryl cAMP (5 mg/ml), both of which increase intracellular cAMP, stimulate apo-E secretion 9-fold and 12-fold, respectively. The dibutyryl cAMP effect is dependent on both dose (greater than or equal to 0.5 mg/ml required) and time (onset at 24 h, maximum at 48 h, and back to near baseline at 96 h). Isobutylmethylxanthine, a phosphodiesterase inhibitor, augments FSH-stimulated apoprotein E synthesis 2.5-fold, supporting a role for cAMP in mediating the FSH effect. This is the first demonstration of the hormonal regulation of apoprotein E synthesis in an extrahepatic tissue.     

Influence of organosulphur compounds from garlic on the secretion of matrix metalloproteinases and their inhibitor TIMP-1 by cultured HUVEC cells

Organosulphur compounds from garlic, especially diallyl disulphide (DADS) at non-toxic concentrations, affected production and secretion of some matrix metalloproteinases (MMPs) and of tissue inhibitor of metalloproteinase-1 (TIMP-1), one of their inhibitors, by human umbilical vein endothelial cells. Addition of DADS to the culture medium resulted in a concentration-dependent reduction of secreted MMP-2 protein and activity as well as TIMP-1 protein. In the presence of inducers (phorbol 12-myristate 13-acetate, forskolin and tumor necrosis factor alpha) addition of DADS caused a distinct concentration-dependent decrease of MMP-9 and TIMP-1 secretion, while not affecting MMP-9 mRNA levels. Intracellular protein levels remained low and were not affected. Other organosulphur compounds like allyl mercaptan and S-allylcysteine showed no or less clear effects on MMP-secretion or TIMP-1-secretion. These results suggest that DADS may mediate some of the biological effects ascribed to garlic preparations through affecting MMP-TIMP balance.     

Combined effect of phorbol ester and, A23187 or dibutyryl cyclic AMP on pepsinogen secretion from isolated gastric glands

In isolated guinea pig gastric glands, pepsinogen secretion was stimulated by the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in a dose dependent manner. Calcium-deprivation from the medium resulted in the decrease in TPA-induced pepsinogen secretion. The combination of 0.4 microM Ca2+ionophore A23187 and TPA stimulated pepsinogen secretion slightly higher than the calculated additive value for each agent. This synergistic effect of the agents supports a role of calcium-activated, phospholipid-dependent protein Kinase (protein Kinase C) in gastric pepsinogen secretion. Furthermore, pepsinogen secretion was also stimulated by dibutyryl cyclic AMP (dbc AMP) and dbc AMP slightly enhanced TPA-induced pepsinogen secretion. Results suggest that gastric chief cells possess at least two different secretory pathways for pepsinogen which are probably dependent on protein kinase C and cyclic AMP, respectively.     

Correlation between NADPH-diaphorase and iNOS in bank vole Leydig cells in vitro and in testicular sections with the use of histochemistry and immunocytochemistry

Histochemistry for NADPH-diaphorase detects an enzymatic activity associated with nitric oxide synthase while immunohistochemistry detects the nitric oxide synthase molecule. NADPH-diaphorase and inducible isoform of nitric oxide synthase in Leydig cells in vitro and in testis sections of the bank vole were demonstrated histochemically and immunocytochemically. Histochemical studies revealed localization of NADPH-diaphorase reaction product in the cytoplasm of cultured Leydig cells as well as in the interstitial area, mainly in Leydig cells and in vascular endothelium. Distribution pattern of NADPH-diaphorase was different in Leydig cell cytoplasm of individual cells. Using immunocytochemistry, the immunoreactivity for nitric oxide synthase was observed both in cultured Leydig cells and testis sections. Moreover, a co-localization of positively immunostained cells with those histochemically detected was noticed. Addition of hCG to the cultured medium or injections in vivo resulted in a small decrease in reaction intensity in Leydig cells. Treatment with N omega-nitro-L-arginine methyl ester resulted in distinctly weaker reactivity of the enzymes studied which was correlated with a higher testosterone and estradiol levels in Leydig cells measured radioimmunologically. The results have indicated that nitric oxide synthase is able to act directly within the male gonad regulating androgen secretion by Leydig cells.     

In vitro responses of luteinizing rat granulosa cells to human thyroid-stimulating hormone

The effect of human thyroid-stimulating hormone (hTSH) on progesterone (P4) secretion during initial luteinization and subsequent prolactin (Prl)-mediated steroidogenesis by cultured rat granulosa cells was studied. Granulosa cells, obtained from pregnant mare's serum gonadotropin (PMSG)-treated immature female rats, were preincubated for 1, 3, 6, 12, or 24 h in control medium lacking added hormones or in medium containing 1.0 microgram/ml human chorionic gonadotropin (hCG) or hTSH, and maintained subsequently for 6 days in medium containing 1.0 microgram/ml bovine (bPrl). Indices of luteotropic stimulation were provided by: 1) elevated P4 concentrations determined by radioimmunoassay of spent media samples; and 2) cytoplasmic lipid accumulation assessed by osmium tetroxide staining following fixation after 7 days of culture. Progesterone levels in media from cultures exposed to hCG for 24 h were twofold higher than control cultures, whereas those in media from cultures preincubated in hTSH for 24 h were fourfold higher than control levels. Cultures preincubated in 1.0 microgram/ml hCG for as little as 1 h and then maintained for 6 days in Prl secreted significantly more P4 than did control cultures also maintained with Prl for 6 days. Cultures preincubated in hTSH required a 24-h exposure before a significant increase in Prl-mediated P4 secretion was observed. Intensity of cytoplasmic osmiophilia correlated directly with P4 concentration. These results suggest that: 1) hTSH has the ability to promote P4 secretion during initial luteinization and to regulate subsequent Prl-mediated steroidogenesis by cultured rat granulosa cells; and 2) the mechanism by which hTSH stimulates Prl-mediated P4 secretion in this model system may differ from that of hCG.     

Differential steroidogenic responses of ovine luteal cells to ovine luteinizing hormone and human chorionic gonadotropin

Experiments were conducted in vitro on ovine small luteal cells to evaluate their steroidogenic response to ovine luteinizing hormone (oLH) and human chorionic gonadotropin (hCG) administered continuously throughout the experimental period or as a 15-min pulse. Both oLH and hCG stimulated a significant increase in progesterone secretion (P less than 0.001) by small luteal cells. Human chorionic gonadotropin administered continuously or as a pulse maintained progesterone secretion at 40-55% of experimental maximum at least 6 hr while oLH-stimulated progesterone secretion declined to basal levels by 4 hr after a 15-min pulse or declined to 25% of the experimental maximum within 6 hr under constant stimulation. The responses of small luteal cells to oLH and hCG were found to differ (P less than 0.001). The sustained progesterone secretion of luteal cells in response to a pulse of hCG may be due to longer residence of occupied receptor complex on the cell membrane. In contrast, the decline in oLH stimulated progesterone secretion, even when hormone is continuously present in the medium, may be related to a rapid internalization of receptor-hormone complexes and down-regulation of receptors.     

Characterization of hCG regulation in cultured human amniotic fluid cells: II. Mechanisms for stimulation

We showed previously that sodium butyrate stimulated human chorionic gonadotropin (hCG) measured by radioimmunoassay of medium from human second trimester amniotic fluid cell cultures, termed AF cells. We now find that stimulation of hCG in the presence of sodium butyrate takes as long as 20 h. When AF cells are preincubated with sodium butyrate, hCG levels increase in direct relation to length of the preincubation period. These findings suggest that elevation of hCG is not due merely to a release of hormone from the cells. Addition of cycloheximide or Actinomycin D inhibited protein synthesis and RNA synthesis, respectively, and prevented the stimulation of hCG by sodium butyrate. These results lend support for a mechanism of regulation involving protein and RNA synthesis, the increase in hCG levels being due to new synthesis of the hormone. Other agents reported to influence hCG production by different types of cell cultures include dibutyryl cyclic AMP, epidermal growth factor (EGF), methotrexate, and hydroxyurea. Dibutyryl cyclic AMP and EGF have no effect on hCG production in our AF cells: methotrexate causes a minimal increase, hydroxyurea causes a further increase, but sodium butyrate has the strongest stimulatory effect. We conclude that amniotic fluid cells in culture are susceptible to environmental agents capable of modulating synthesis of hCG by mechanisms involving synthesis of RNA and protein.