Acute stimulation of human chorionic gonadotropin secretion by recombinant human activin-A in first trimester human trophoblast.

Inhibin and activin are synthesized by the placenta, raising questions as to their functions in this tissue. The possibility of local regulation of other placental hormones critical during pregnancy was investigated by examining the effects of recombinant human inhibin-A and activin-A on hCG secretion. Potential interactions with GnRH-stimulated hCG secretion were also explored. First trimester human placental tissue was physically dissociated to isolate trophoblast cells. Cells were cultured on microcarrier beads for 7-10 days and loaded into 1.5-ml chambers in a perifusion system. After a 1-h control period, hormone treatments were administered in the perifusion medium for 5 min, and perifusate was collected for an additional 55 min. Perifusate fractions were analyzed for hCG concentration by RIA. Both activin and GnRH stimulated a rapid and transient hCG secretory response in the perifusion system. Unlike the effect of GnRH, the activin-stimulated hCG response was not blocked by concomitant treatment with a GnRH antagonist. The hCG RIA results were confirmed by preliminary experiments using a novel hCG bioassay. This suggested that activin did not facilitate hCG secretion by stimulation of endogenous GnRH release. Inhibin alone did not affect hCG secretion or GnRH-stimulated hCG secretion. However, treatment with inhibin and activin in combination resulted in a transient increase in hCG, followed by a decrease in hCG secretion to below pretreatment levels. The results suggested that in addition to GnRH, activin may play a role in the regulation of hCG secretion in first trimester placenta.

Characterization of cell types within a chorionic gonadotropin-secreting, mechanically dissociated human placental cell population in perifusion.

A cell perifusion system was established to examine human placental endocrine regulation by locally synthesized peptides. First-trimester and term trophoblast cells were mechanically dissociated. Cells were plated on microcarrier beads and cultured for 7-14 days. Cells on beads were loaded in chambers, perifused with culture media and effluent was assayed for chorionic gonadotropin (hCG). Mechanical dissociation of placental tissue produced cell preparations with 85-95% viability. Staining with Masson trichrome, cytokeratin and beta-hCG antibodies suggested that greater than 50% of the cells were trophoblast. Perifused trophoblast cells secreted hCG in a continuous non-pulsatile fashion, independent of exogenous hormonal stimuli. hCG secretion from first-trimester trophoblast cells remained stable in static culture for 14 days. GnRH perifusion (10(-8) M) for 15-120 s transiently increased hCG secretion from first-trimester trophoblast cells. Longer GnRH exposure stimulated greater hCG secretion. Each of 3 consecutive pulses of 8-bromo-cyclic adenosine-3',5'-monophosphate (cAMP, 10(-9) M) administered at 2-hour intervals stimulated transient hCG secretion from first-trimester and term placental cells. cAMP stimulated hCG secretion more potently from first-trimester than from term placental cells.

Rapid stimulation of human chorionic gonadotropin secretion by interleukin-1 beta from perifused first trimester trophoblast.

Placental trophoblast has been implicated as a major source of interleukin-1 beta (IL-1 beta), a cytokine that mediates immunological responses in the body. This study evaluated the effect of IL-1 beta on hCG secretion from 8- to 12-week-old placental trophoblast. Physically dissociated trophoblast cells collected from multiple placentae were cultured on carrier beads and loaded into chambers in a perifusion system. Medium was perifused through the chambers, and effluent was collected and assayed for hCG. Basal hCG secretion was not dependent on exogenous IL-1 beta or GnRH, but varied between mixed placental preparations and increased with duration of culture. Therefore, hCG secretion was expressed as a percentage of mean basal hCG secretion for any given chamber. IL-1 beta (10(-9) M) stimulated a rapid and transient hCG secretory response. hCG release increased by approximately 150% (P less than 0.05; n = 5) in response to the cytokine, but lower concentrations (10(-10) and 10(-11) M) were ineffective (P greater than 0.05; n = 3 each). GnRH stimulated hCG secretion by approximately 80% (P less than 0.05; n = 6). The hCG secretory profiles in response to IL-1 beta and GnRH were similar. Combined treatment with equimolar (10(-9) M) IL-1 beta and GnRH increased hCG secretion by approximately 150% (P less than 0.05; n = 5), stimulating hCG secretion as effectively as either hormone alone (P greater than 0.05). The stimulatory effect of GnRH on hCG secretion was blocked by the concomitant presence of a GnRH antagonist, Nal-Glu-GnRH (P less than 0.05; n = 5). However, simultaneous treatment with IL-1 beta and Nal-Glu-GnRH did not affect IL-1 beta-stimulated hCG secretion (100.5 +/- 3.6 vs. 162.9 +/- 10.2%; P less than 0.05; n = 7). The data suggest that IL-1 beta and GnRH stimulated a near-maximal physiological hCG secretory response, possibly through different receptor types. Alternatively, these two hormones may share a common signal transduction pathway, or IL-1 beta may influence a step distal to the coupling of GnRH to its receptor in the placental trophoblast.

Intracellular calcium and the signaling mechanism of luteinizing hormone-releasing hormone in rat granulosa cells.

OBJECTIVE: The purpose of these studies was to determine the source(s) of the increase in intracellular free calcium in response to luteinizing hormone-releasing hormone in ovarian granulosa cells. STUDY DESIGN: Rat granulosa cells were cultured and loaded with fura-2-acetoxy-methyl ester, a fluorescent calcium indicator dye, and intracellular free calcium measured by microspectrofluorometry. The source of the luteinizing hormone-releasing hormone induced increase in intracellular Ca++ was investigated with various calcium channel blockers (verapamil, diltiazem, and nifedipine), high K+ buffer, and perifusion with media lacking Ca++. RESULTS: All three voltage-sensitive calcium channel blockers (10(-5) mol/L) tested were ineffective in blocking the luteinizing hormone-releasing hormone induced intracellular Ca++ increase. Treatment with high K+ buffer also had no effect. Perifusion with media lacking calcium resulted in a gradual loss of the luteinizing hormone-releasing hormone response, an effect that was accelerated by repeated stimulation with hormone. Transient replacement of extracellular Ca++ failed to restore the response but continued perifusion with Ca(++)-replete media allowed a luteinizing hormone-releasing hormone response 10 minutes later. CONCLUSIONS: The luteinizing hormone-releasing hormone induced intracellular Ca++ increase does not appear to result from the opening of voltage-sensitive or K(+)-dependent Ca++ channels. To the contrary, this response likely results from the release of Ca++, primarily from intracellular stores.

Intracellular signaling in the gonads.

The regulation of steroidogenesis in both the ovary and testis involves a complex interaction of a diversity of hormones and intracellular signaling pathways. The recent cloning of LH and FSH receptors has paved the way for an increased understanding of the mechanisms of receptor conformation, ligand-receptor interaction, and facilitation of post-receptor activity. The dominant role played by LH in the regulation of steroid production appears to be mediated by more than one intracellular signaling pathway. In addition to the stimulation of the adenylate cyclase-cAMP pathway, also known to be stimulated by FSH, the actions of LH may be additionally mediated by other intracellular messengers, such as those derived from the PLC pathway. Steroidogenesis in the gonads appears to be modulated by a variety of factors in addition to the gonadotropins. In this review, those factors of intracellular signaling mechanisms of which we have some understanding have been discussed. These include GnRH, PGF2 alpha, Ang II, VIP, GHRH, TNF alpha, CRF, EGF, and TGF alpha. Many of these factors have been shown to be locally synthesized, and specific receptors have been identified in the gonads. Many gonadal factors have the capacity to exert effects on steroidogenesis independent of the gonadotropins. Alternately, they have been demonstrated to alter the gonadal response to the gonadotropins via autocrine, paracrine, and intracrine mechanisms. As yet, our understanding of the intracellular signaling mechanisms used by novel gonadal regulators is limited. The involvement of the PLC, PLA2, and PLD pathways in this regard has been reviewed. It is becoming apparent that multiple signaling pathways may be stimulated by a single hormone, as in the case of GnRH, PGF2 alpha, and LH. The complexity of intracellular signal transduction in the gonads is enhanced by the potential cross-talk at numerous steps in the signaling cascades.

Luteinizing hormone-releasing hormone (LHRH)- and (hydroxyproline9)LHRH-stimulated human chorionic gonadotropin secretion from perifused first trimester placental cells.

(Hydroxyproline9)LHRH [(Hyp9)LHRH] has been isolated from human, sheep, rodent, and frog hypothalamus. (Hyp9)LHRH is the major LHRH moiety in fetal rat hypothalamus. This study compared 1) synthetic LHRH-stimulated hCG secretion from term trophoblast vs. first trimester placental cells and 2) the ability and specificity with which synthetic LHRH and (Hyp9)LHRH could stimulate hCG secretion from 8- to 12-week gestation placenta. Physically dissociated cells from multiple placentae were pooled, plated on microcarrier beads, and perifused in 1.5-ml chambers (1.5 x 10(6) cells/chamber). Effluent fractions were analyzed for hCG. Each chamber was its own control. Basal hCG secretion did not depend upon exogenous LHRH stimulation. The amplitude of LHRH-stimulated hCG pulses was greater from first trimester placental than term trophoblast cells (mean +/- SEM, 6.99 +/- 1.47 vs. 0.50 +/- 0.05 mIU/ml perifusate; peak minus basal; n = 4 chambers; P less than 0.01). LHRH and (Hyp9)LHRH (10(-9) M) increased hCG secretion from first trimester placental cells (5.02 +/- 1.29 vs. 8.64 +/- 1.61 and 4.36 +/- 0.58 vs. 7.44 +/- 1.01 mIU/ml; n = 15 and 9, respectively; P less than 0.01). At the concentrations used, LHRH and (Hyp9)LHRH seemed to stimulate hCG secretion equipotently (P greater than 0.05). Simultaneous perifusion with an LHRH antagonist, (Nal-Glu)LHRH blocked the hCG secretory response to LHRH or (Hyp9)LHRH (equimolar 10(-9) M concentrations; n = 5; P less than 0.05). (Nal-Glu)LHRH alone (10(-9) M) did not affect hCG secretion (n = 5; P greater than 0.05). The results suggested that first trimester placental cells are more responsive to LHRH than are term trophoblast cells. (Hyp9)LHRH is a potential physiological secretagogue of hCG.

Intragonadal signalling mechanisms in the control of steroid hormone production.

In the gonads, there are two recognized signal transduction mechanisms which operate in the processing of hormonal stimuli. The gonadotropins, follicle stimulating hormone and luteinizing hormone, act primarily through the generation of cyclic AMP. Several other hormonal regulators in the ovary and the testis, such as gonadotropin releasing hormone and prostaglandin F2 alpha stimulate inositol lipid metabolism following receptor binding. This triggers a cascading mechanism which ultimately results in the generation of increased cytosolic free calcium levels, enhanced protein kinase C activity, and liberation of arachidonic acid. There is also evidence that luteinizing hormone shares in the activation of this pathway. In this review, the significance of these signal transduction pathways is discussed in relation to the effects of various hormones on steroid biosynthesis in the gonads.

Prostaglandin F2 alpha and gonadotropin-releasing hormone increase intracellular free calcium in rat granulosa cells.

Changes in cytosolic free calcium concentration ([Ca2+]i) in response to prostaglandin F2 alpha (PGF2 alpha) and gonadotropin-releasing hormone (GnRH) were measured in single rat granulosa cells, using the calcium-sensitive fluorescent dye, fura-2AM. In 90 out of 135 granulosa cells (67%), there was a 3- to 4-fold increase in resting [Ca2+]i within 30 s of administration of PGF2 alpha (10(-6) M). The resting [Ca2+]i returned to pre-stimulation levels in approximately 80 s. Granulosa cells were responsive to PGF2 alpha at concentrations ranging from 10(-7) M to 10(-4) M (n = 7). Within this range of concentrations, the magnitude of the calcium response did not differ. In another series of experiments, the majority (93%, n = 57) of the granulosa cells which responded to PGF2 alpha also responded to GnRH. Neither the magnitude of the [Ca2+]i response nor the time to response differed between PGF2 alpha and GnRH. Furthermore, simultaneous treatment of granulosa cells with both hormones did not generate a larger response than with either hormone alone. During perifusion with low calcium media, the characteristic [Ca2+]i response to PGF2 alpha decreased, and was eliminated within 10 min (n = 9). Similar observations were made in response to GnRH under these conditions. These data confirm that PGF2 alpha and GnRH stimulate a transient increase in [Ca2+]i in rat granulosa cells, the source of which may be shared intracellular stores.