Elevated levels of homocysteine produce detrimental effects in humans but its role in preterm birth is not known. Here we used a mouse model of hyperhomocysteinemia to examine the relevance of homocysteine to preterm birth. The mouse carries a heterozygous deletion of cystathionine β-synthase (Cbs(+/-)). Gestational period was monitored in wild type and Cbs(+/-) female mice. Mouse uterine and placental tissues, human primary trophoblast cells, and human myometrial and placental cell lines were used to determine the influence of homocysteine on expression of specific genes in vitro. The activity of BKCa channel in the myometrial cell line was monitored using the patch-clamp technique. We found that hyperhomocysteinemia had detrimental effects on pregnancy and induced preterm birth in mice. Homocysteine increased the expression of oxytocin receptor and Cox-2 as well as PGE2 production in uterus and placenta, and initiated premature uterine contraction. A Cox-2 inhibitor reversed these effects. Gpr109a, a receptor for niacin, induced Cox-2 in uterus. Homocysteine upregulated GPR109A and suppressed BKCa channel activity in human myometrial cells. Deletion of Gpr109a in Cbs(+/-) mice reversed premature birth. We conclude that hyperhomocysteinemia causes preterm birth in mice through upregulation of the Gpr109a/Cox-2/PGE2 axis and that pharmacological blockade of Gpr109a may have potential in prevention of preterm birth.

Introduction: Mice with a heterozygous deletion of the enzyme cystathionine-b-synthase (Cbs) have increased plasma levels of homocysteine and deliver two days earlier than wild-type mice. This process is associated with increased expression of cyclooxygenase-2 (Cox-2) and oxytocin in the uterus and placenta. However, both the administration of oral Cox-2 inhibitors to pregnant Cbs+/- mice prior to their anticipated delivery date and the deletion of GPR109A reverse this process, suggesting that this G-protein-coupled receptor regulates Cox-2 expression. Our aim was to investigate the effects of hyperhomocysteinemia on premature delivery through Cox-2 and oxytocin expression and its reversal through oral Cox-2 inhibitors and the deletion of GPR109A. Methods: Placental and uterine tissue was obtained from Cbs+/- female mice on the 16th day of gestation and compared to wild-type females of the same gestational day (Cbs+/+). Cox-2 and oxytocin levels were assessed by qPCR, western blot, and immunohistochemistry. in vitro studies were performed by measuring uterine muscle contraction. to reverse premature delivery, a Cox-2 inhibitor was administered orally for two days prior to the anticipated delivery date. to study the effects of GPR109A on premature delivery, Cbs+/-/Gpr109a-/- mice were obtained by crossing Cbs+/- mice and Gpr109a-/- mice, and the gestational period was compared between Cbs+/-/Gpr109a+/+ mice and Cbs+/-/Gpr109a-/- mice. Results: Cox-2 and oxytocin expression were significantly increased in the Cbs+/- pregnant females compared to pregnant wild-type females. These results were confirmed by qPCR, western blot, and immunohistochemistry. Oral administration of a known Cox-2 inhibitor to Cbs+/- females resulted in the reversal of premature delivery with a total gestation period similar to wild-type mice. GPR109A deletion also reversed premature delivery. Cbs+/-/Gpr109a+/+ mice delivered on gestational day (GD) 16.6 ±0.12 whereas Cbs+/-/Gpr109a-/- mice and wild-type mice delivered pups on GD 20 ±0.5 and 20 ±0.17, respectively. Additionally, overall expression of Cox-2 and oxytocin were lower in Cbs+/-/Gpr109a-/- mice, further supporting the idea that Cox-2 acts through GPR109A. Conclusions: Elevated levels of homocysteine cause premature delivery in mice through increased expression of Cox-2, which we believe acts through a G-protein coupled receptor called GPR109A. These Cbs+/- females have increased plasma levels of homocysteine and demonstrate increased expression of Cox-2 and oxytocin in their uterus and placenta, which supports the idea of increased contraction leading to premature delivery. Gpr109A is a niacin-dependent receptor that is obligatory for this process and has been proven to facilitate increased Cox-2 expression in human dendritic cells. We therefore conclude that homocysteine-induced premature delivery in Cbs+/- mice is mediated through Gpr109A and its deletion reverses the premature delivery process, as does oral treatment with Cox-2 inhibitors.

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It has been found that besides the meroterpene bakuchiol, first reported in 1966 in the pericarp of seeds of Psoralea corylifolia, three oxygenated derivatives also occur, and these have been identified as 2,3-epoxybakuchiol, Delta(1),3-hydroxybakuchiol, and Delta(3),2-hydroxylbakuchiol.

By using four different cell isolation procedures, we previously identified two morphologically and biochemically distinct Leydig cell populations in rat testis. The light cells were vacuolated and bound 125I-labeled human choriogonadotropin (hCG) with high affinity but upon hCG stimulation in vitro, cAMP and testosterone production by these cells were minimal. On the other hand, the heavier cells displayed typical Leydig cell morphology and bound very little hCG but vigorously produced cAMP and testosterone (Browne, E.S., Bhalla, V.K., 1991, J. Androl. 12:132-139). This study examines the distribution of LH/hCG receptor mRNAs in the two cell types. The light cell fraction contains larger transcripts of LH/hCG receptor but the heavier Leydig cells contain shorter transcripts. The observations raises the intriguing possibility that shorter rather than larger LH/hCG receptor transcripts are responsible for the induction of a biologically functional, G-protein coupled, LH/hCG receptor in Leydig cells.

Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10−10m) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable 125I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 × 106 cells/250 μl) were used for in vitro hCG stimulation assays. Under these conditions, the 125I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.

The dose-response relationship between luteinizing hormone/human chorionic gonadotropin (LH/hCG)-stimulated biological response and 125I-labeled hCG binding was studied in purified Leydig cells from adult rat testes. The concentration of hCG needed for one-half maximal stimulation of cyclic adenosine monophosphate (cAMP) and testosterone production (ED50) was 2.16 x 10(-11)mol/L and 5.6 x 10(-13)mol/L, respectively. This suggests that extremely low levels of hormone in the range of 10(-13)mol/L hCG are sufficient to generate enough cAMP (5.66 pmol; 2.83 x 10(-9)mol/L) for steroidogenesis, thereby preserving the catalytic potential of the receptor-cyclase system. Most of the cAMP formed at 10(-13)mol/L hCG was released into the medium, and the intracellular cAMP was much less and barely detectable (0.98 x 10(-9)mol/L; 1.96 pmol/2 x 10(6) cells). The specific binding of 125I-labeled hCG to purified Leydig cells at a correspondingly higher hCG concentration (3 x 10(-10)mol/L) was extremely low and did not display a dose-dependent increase in binding. Assuming the specific binding to represent 100% occupancy of high affinity receptors (14.2 fmol/2 x 10(6) cells per 2 ml), each mole of bound hCG generated 15,423 mol cAMP and 12,817 mol testosterone. The results show that the hormone interacts with cellular receptors as a catalyst to generate the biological response. Moreover, the true affinity of hormone-receptor interaction responsible for the physiologic action is possibly much greater than previously reported for this system. This information should prove useful for reconstitution studies using the hormone receptor/G-protein/adenylate cyclase system in vitro in soluble form.

The JAR human placental choriocarcinoma cell line transports serotonin, accumulating the monoamine inside the cell against a concentration gradient. The transport is energized by an NaCl gradient. Tricyclic (imipramine and desipramine) and non-tricyclic (paroxetine and fluoxetine) antidepressants inhibit the transporter markedly, but reserpine and 5-hydroxytryptophan do not. Ouabain, gramicidin, and nigericin, which reduce or abolish the transmembrane Na+ gradient, and phloridzin, which interferes with glucose transport into the cells, inhibit the transport. Preincubation of the cells with glucose-free medium also causes similar inhibition. The activity of the serotonin transporter in this cell line is stimulated in response to overnight (16-h) incubation with increasing concentrations of cholera toxin (0.1-1,000 ng/ml). Under these conditions the stimulation is maximal at 10 ng/ml cholera toxin (3.1 +/- 0.2-fold). Cholera toxin increases the cAMP content of these cells by several hundredfold within 2 h. Isobutylmethylxanthine (100 microM), dibutyryl cAMP (100 microM), and forskolin (100 microM) mimic the action of cholera toxin, eliciting a 1.6-2.5-fold stimulation of the serotonin transporter activity. The stimulatory effect of cholera toxin is antagonized significantly by simultaneous incubation of the cells with 50 microM N-(2-aminoethyl)-5-isoquinolinesulfonamide, a protein kinase inhibitor. The effect of cholera toxin on serotonin transport is specific because, under similar conditions, cholera toxin inhibits 3-O-methyl-D-glucose transport and does not influence taurine transport in this cell line. There is also no significant change in the protein content of the cells after cholera toxin treatment. Kinetic analysis reveals that cholera toxin causes an increase in the maximal velocity (7.89 +/- 0.67 to 17.55 +/- 1.06 pmol/mg of protein/5 min) and a decrease in the Michaelis-Menten constant (0.52 +/- 0.09 to 0.29 +/- 0.04 microM). These data show that the JAR human placental choriocarcinoma cell line expresses a high affinity serotonin transporter that is sensitive to inhibition by antidepressants and that the activity of the transporter is under cAMP-dependent regulation.

Rat testicular interstitial cells have been separated by discontinuous/continuous gradient of Percoll, yielding four cell fractions. The light cells in fraction I bound luteinizing hormone/human chorionic gonadotropin (LH/hCG) with high affinity but were not steroidogenic in response to hormone. Fraction II consisted mainly of germ cells. Although fraction III contained Leydig cells, this fraction was contaminated with germ cells and was less responsive to hormone as compared to the Leydig cells in fraction IV. The Leydig cells in fraction IV produced cAMP and testosterone in response to hormone action in a manner which was critically dependent upon cell concentration. The production of cyclic adenosine monophosphate (cAMP) in the presence of saturating concentrations of hCG (2.4 × 10t1̄0 M) was linear as a function of cell concentration up to 7.0 × 106 cells/1.25 ml and thereafter, a slight inhibition (26%) was seen at 10 × 106 cells/1.25 ml. The average value for cAMP production by hCG was 133.8 ± 8.5 pmol cAMP/2 × 106 cells. The production of testosterone was biphasic, increasing linearly up to 5 X 106 cells/1.25 ml and decreasing thereafter. Two million cells, in the presence of 2.4 × 10−10 M hCG, produced an average of 24.2 ± 1.7 ng of testosterone in reaction volumes ranging from 1 to 2 ml whereas the same number of cells only produced 5.1 ± 0.6 ng of testosterone in 250 μ1. The binding of 125I-labeled hCG to the same batch of cells increased with increasing cell concentrations as expected but under the conditions of maximal steroidogenesis at low cell concentrations (1.25 2.0, and 2.5 × 106 cells/1.25 ml), it was barely detectable. Thus, we conclude that there is an inverse relationship between the parameters of binding and biological response in purified Leydig cells.

Two human chorionic gonadotropin (hCG) responsive cells from rat testicular interstitium were previously isolated on a discontinuous gradient of Percoll. The light cells were non-steroidogenic and bound 125I-labeled hCG with high affinity (Kd 3.0 x 10(-10) mol/L), whereas the steroidogenic heavier cells (Leydig cells) produced cyclic adenosine monophosphate (cAMP) and testosterone in response to hCG stimulation with very little hCG binding. In that study, the heavier cell fraction was contaminated with germ cells, red blood cells, and other cells. These cells have now been further purified on a continuous gradient of Percoll (20 to 60%, v/v), and have resolved into three visible bands. The cells in subfraction I, predominantly damaged Leydig cells, germ cells, and/or residual light cells, bind 125I-labeled hCG with high affinity (Kd 4.09 x 10(-10) mol/L) without producing cAMP and testosterone in response to hCG. Subfraction III consists mainly of red blood cells. The cells in subfraction II, identified as typical Leydig cells by electron microscopy, produce cAMP and testosterone in response to hCG but, again, bind only a small amount of hCG (4.5 +/- 0.3 fmol/2 x 10(6) cells/250 microliters/per hour at 37 degrees C). Thus, further purification of the heavier cell fraction from a discontinuous gradient of Percoll on a continuous gradient of Percoll yields Leydig cells, free of contaminating germ cells and red blood cells, which actively produce cAMP and testosterone with a very low level of hCG binding, the affinity of which is undetectable by current binding techniques.

In order to determine the significance of carbohydtrate residue of human chorionic gonadotropin )hCG) in receptor interaction and signal transduction leading to steriodogenesis, the effect of deglycosylated hCG (DG-hCH) was studied in vitro with two different hCG-responsive purified testicular interstitial cell fractions. Fractions I light cells, previously found to bind 125I-labeled hCG with high affinity witout producing testosterone, also bound 125I-labeled DG-hCG with high affinity (Kd 7.2·10−1) M) wihtout stimulating testosterone production. Fraction IV heavier cells, which produced testosterone in response to hCG without detectable high-affinity hCG-binding sites, neither bound DG-hCG nor sufficiently produced cAMP and testosterone in response. With the addition of intact hCG, DG-hCG inhibited cAMP levels, although not sufficiently to inhibit testosterone production. This observation was contrary to previous studied in which DG-hCG was shown to be antagonist to hCG action. We conclude that: (a) DG-hCG retains its binding activity in light cells and this high-affinity binding is unrelated to steroidogenesis; (b) DG-hCG does not bind to heavier cells with high affinity and loses its biological activity as results of deglycosylation; (c) DG-hCG actions in this study strengthen the concept of two different hCG-responsive cells in the rat interstitium which, if not separated, will yield misleading data supporting the coexistence of hCG high-affinity binding and biological responses in the same cell; and (d) DG-hCG partially anatagonizes the activation of adenylate cyclase but does not block testosterone production, thus questioning the usefulness of this analogue in antagonizing the action of native hCG in rat testis.

The ability of 125I-labeled human chorionic gonadotropin (125I-labeled hCG) to bind and stimulate steroidogenesis was studied in light cells (density, 1.053-1.065 g/cm3) and heavier cells (density, 1.090-1.110 g/cm3) purified from collagenase-dispersed rat testicular interstitial cells by unit gravity sedimentation (Bhalla, V.K., Rajan, V.P., Burgett, A.C., and Sohal, G.S. (1987) J. Biol. Chem. 262, 5313-5321). Preferential localization of gonadotropin binding sites was demonstrated on light cells, and the heavier cells produced testosterone in response to hCG without occupancy of high affinity (Kd = 2.02 X 10(-10) M) binding sites. In this study, established methods for interstitial cell purification involving gradient centrifugation were utilized to demonstrate the cell heterogeneity. Light cells bound hCG with high affinity (Kd = 3 X 10(-10) M) without manifestation of steroidogenic response. The heavier cells responded to hCG with elicitation of steroidogenesis, but the occupancy was negligible. Stimulation of steroidogenesis by hCG in heavier cells was dose and time dependent. Dibutyryl and bromo cyclic AMP (1 mM) also promoted steroidogenesis comparable to a level stimulated by the tropic hormone (700% stimulation). The concept of spare receptors was tested in purified cell fractions. Upon cell purification, no saturable high affinity binding sites were observed in the heavier cell fraction. Autoradiographic analyses at the electron microscopical level supported this conclusion. Our data suggest that target cell activation is not preceded by hormone occupancy of high affinity binding sites. A model for defining the functional domains of the physiological receptor for hCG is presented.

Two testicular interstitial cell fractions, light and heavier, biochemically and morphologically distinct were obtained by a unit gravity sedimentation procedure. Binding sites for 125I-labeled human chorionic gonadotropin (hCG) were preferentially localized in the light cell fraction (apparent Kd = 2.02 X 10(-10) M; Bmax = 1.17 X 10(-5) nmol/2 X 10(6) cells). These cells did not synthesize testosterone in response to hCG, but the basal release of testosterone was higher than by cells in the heavier fraction (2.49 +/- 0.02 ng/2 X 10(6) cells in the light versus 0.22 +/- 0.00 ng/2 X 10(6) cells in the heavier fraction). The cells in the heavier fraction bound little or no hCG. The binding data from this fraction did not obey saturation kinetics, but testosterone levels were elevated 700-800% in the presence of hCG (i.e. basal value 0.22 +/- 0.00 ng/2 X 10(6) cells versus 1.81 +/- 0.04 ng/2 X 10(6) cells in hCG-stimulated cells). Electron microscopy revealed that heavier cells had features typical of Leydig cells such as large ovoid nucleus with peripherally located heterochromatin, numerous mitochondria with tubular cristae, some lipid droplets, extensively developed smooth endoplasmic reticulum, and well developed Golgi complex. The cells in the light fraction contained an ovoid nucleus with one or more deep infoldings, and their most notable cytoplasmic feature was the presence of numerous vacuoles of varying sizes and shapes. Based upon this and the investigation which follows (Bhalla, V.K., Flasch, M.V., Browne, E.S., Sohal, G.S., and Sharawy, M.M. (1987) J. Biol. Chem. 262, 5322-5332), we conclude that occupancy of high affinity hCG binding sites, generally assumed to be coupled to steroidogenesis, is not necessarily related to the elicitation of this biological response.

The mechanism by which luteinizing hormone (LH) promotes the production of testosterone in Leydig cells by binding to its high affinity sites was reinvestigated. Collagenase dispersed interstitial cells when purified by the application of a variety of techniques such as unit gravity sedimentation, gradient centrifugation, and a combination of the two procedures, were separated into two LH/hCG responsive cell fractions. The two types of interstitial cells displayed distinct biochemical and morphological characteristics. One cell type (the light cell) bound 125I-labeled human chorionic gonadotropin (125I-labeled hCG) with high affinity (Ka approximately equal to 3.33 x 10(9) M-1) but testosterone was not produced by this cell type as a result of hCG target cell receptor interaction. On the other hand, hCG stimulated the production of testosterone in another cell type (the dark/heavier cell). Steroidogenesis was maximally stimulated (700-800 percent over basal) by concentrations of hCG in the range of 3 x 10(-10) M, but high affinity binding sites for 125I-labeled hCG were not detectable. The residual binding that occurred did not obey saturation kinetics and was predominantly nonspecific. The stimulation of steroidogenesis by hCG in dark/heavier cells was dose and time dependent. Addition of dibutyryl or bromo cAMP (1 mM) to the cell suspension resulted in production of testosterone demonstrating the involvement of an hCG sensitive adenylate cyclase system in the transfer signaling process. These observations suggest the lack of a direct association between the occupancy of high affinity binding sites by hCG and testosterone production in rat Leydig cells. The stimulation of a biological response by a pathway independent of hCG occupancy of high affinity binding sites on Leydig cell is discussed and morphology of light and dark/heavier cells is presented. Autoradiographic evidence substantiates the conclusions.

Alcohol: Clinical and Experimental ResearchVolume 7, Issue 2 p. 153-162 Alcohol-Induced Luteinizing Hormone Receptor Deficiency at the Testicular Level Vinod K. Bhalla PhD, Corresponding Author Vinod K. Bhalla PhD Department of Endocrinology, Medical College of Georgia, Augusta, GA.2 Department of Endocrinology, Medical College of Georgia, Augusta, GA 30912.Search for more papers by this authorValanila P. Rajan PhD, Valanila P. Rajan PhD Department of Endocrinology, Medical College of Georgia, Augusta, GA.Search for more papers by this authorMeredith E. Newman BS, Meredith E. Newman BS Department of Endocrinology, Medical College of Georgia, Augusta, GA.Search for more papers by this author Vinod K. Bhalla PhD, Corresponding Author Vinod K. Bhalla PhD Department of Endocrinology, Medical College of Georgia, Augusta, GA.2 Department of Endocrinology, Medical College of Georgia, Augusta, GA 30912.Search for more papers by this authorValanila P. Rajan PhD, Valanila P. Rajan PhD Department of Endocrinology, Medical College of Georgia, Augusta, GA.Search for more papers by this authorMeredith E. Newman BS, Meredith E. Newman BS Department of Endocrinology, Medical College of Georgia, Augusta, GA.Search for more papers by this author First published: March 1983 https://doi.org/10.1111/j.1530-0277.1983.tb05431.xCitations: 16AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume7, Issue2March 1983Pages 153-162 RelatedInformation

By using pair-feeding technique, the number of gonadotropin binding sites per unit mass of testicular homogenate was measured 15, 30, 45 and 60 days after ethanol administration to 60-day-old male rats. The ingestion of ethanol (5%, v/v) as a part of a nutritionally adequate liquid diet resulted in 30, 35 and 40% reduction in gonadotropin receptors by 15, 30 and 45 days, respectively. The body weights in the alcohol-fed rats were comparable to pair-fed controls. These findings suggest that the presence of ethanol in the liquid diet depresses the level of gonadotropin receptors in the rat testis.

The effects of LH, ethanol, and acetaldehyde on the in vitro release of cAMP into the medium from decapsulated rat testes were studied, and the data were correlated with other measured parameters, such as the total tissue cAMP and the binding of 125I-labeled hCG to testicular receptors. The concentration of cAMP in the medium after incubation of the testes with low concentrations of LH (50 ng/testis) for 2 h at 27 C was found to be 81.6 ± 3.8 pmol/testis compared to 43.6 ± 2.9 pmol in controls (an 87% stimulation). The release of cAMP from the testis was not accompanied by a detectable decrease in 125Ilabeled hCG binding as predicted by the occupancy theory of hormone receptor interactions. Tissue levels of cAMP were maintained at 100 ± 2.5 pmol/testis. High concentrations of hormone did not further increase the release of cAMP, but the receptor sites were decreased. Treatment of decapsulated rat testes with ethanol (10%, vol/vol) under similar experimental conditions resulted in a 71% increase in cAMP levels in the medium. The tissue cAMP levels were also elevated in test groups. Binding patterns were comparable to those in controls. FSH and acetaldehyde had no effect on any of the above parameters. High concentrations of ethanol (>10%) decreased the number of receptors, with a loss of biological response. Kinetic experiments revealed that maximal stimulation of cAMP production (115 ± 12 pmol/testis) by 10% ethanol occurred 30 min after incubation. The tissue cAMP levels and the binding sites for hCG did not substantially differ from those values reported in previous experiments with ethanol. On the basis of the present observations, it is suggested that hormone and ethanol activate the tissue by altering the characteristics of the membrane; receptor occupancy may not be required for biological response.

After a single i.p. injection of human chorionic gonadotropin (hCG) (CR 121;6.4 μg/animal), the number of gonadotropin receptors in the rat testis has been found to increase as a function of time. The number of receptors in the rat testis was maximal at 3 h and remained elevated for at least 5 to 6 h before dropping to values below nontreated control samples. During this period of intense activity in the testis, the basal cyclic AMP (cAMP) and testosterone levels were increased. Despite high levels of biologically and immunologically active hCG in circulation, the receptor occupancy was minimal [<5% of the apparent number of binding sites (Bmax)]; the ability of the intact testis to produce/release cAMP into the medium in response to minute quantities of added human luteinizing hormone (hLH) in vitro was greater. At 3 h, soluble receptors accumulated in the interstitial fluid and the testis was highly vascular. These results suggest that the levels of receptors fluctuate in the testis, and their levels in the tissue dictate the biologic response.