Action of the opioid agonist FK 33-824 on porcine small and large luteal cells from the mid-luteal phase: effect on progesterone, cAMP, cGMP and inositol phosphate release.

The present study was designed to investigate the effect of the opioid agonist FK 33-824 on basal and hCG-induced progesterone (P4), cAMP and cGMP secretion and on the phosphoinositide-specific phospholipase C signalling system in separated porcine small (SLCs) and large luteal cells (LLCs). Unit gravity sedimentation was used to produce cultures of small and large luteal cells from corpora lutea (CL) on days 8-10 of the oestrous cycle. In order to examine the effect of FK 33-824 on P4 and cyclic nucleotide release, SLCs and LLCs were incubated in M199 medium at 37 degrees C in 5% CO2:95% air, for 12 h. Small and large luteal cells were treated with hCG (100 ng/ml) alone, FK 33-824 (10(-9) M) alone or were co-treated with FK 33-824 and hCG and with the opioid antagonist, naloxone (NAL, 10(-5) M). FK 33-824 alone did not influence P4 secretion by LLCs and SLCs. However, FK 33-824 completely abolished the stimulatory effect of hCG on P4 secretion by SLCs. The addition of FK 33-824 was followed by a significant increase in cAMP release (p<0.01) by LLCs and a decrease in cGMP secretion by SLCs (p<0.05). The effect of FK 33-824 was blocked by NAL, which strongly suggests that the observed influence of this opioid agonist was achieved through its binding to opioid receptors in luteal membranes. In the presence of hCG, cAMP secretion by both SLCs and LLCs was many-fold higher than in the control group. As regards cGMP output, only LLCs showed elevated secretion of this cyclic nucleotide under the influence of hCG. With the aim of examining the influence of FK 33-824 on phosphatidylinositol hydrolysis, LLCs, SLCs and mixed small and large cells were labelled with [3H]-myo-inositol (100 microCi/ml) for 3 h at 37 degrees C. The cells were then incubated in M199 medium supplemented with 10 mM LiCl, 1% BSA, and antibiotics in the presence and absence of FK 33-824 (10(-9) M) at 37 degrees C for 30 min. Liberated labelled inositol mono-, bis-, and trisphosphates (IPs) were isolated and quantified by affinity chromatography on columns of AG 1-X8 resin, followed by liquid scintillation spectroscopy. Inositol phosphate accumulation in LLCs, SLCs, and mixed small and large cells was not altered by treatment with FK 33-824 at the dose used. In view of these findings we suggest that opioid peptides affect pig corpus luteum steroid secretion, and the response is probably mediated through cyclic nucleotides, but not IPs.     

Effects of selective protein kinase c isozymes in prostaglandin2alpha-induced Ca2+ signaling and luteinizing hormone-induced progesterone accumulation in the mid-phase bovine corpus luteum

A single-cell approach for measuring the concentration of cytoplasmic calcium ions ([Ca(2+)](i)) and a protein kinase C-epsilon (PKCepsilon)-specific inhibitor were used to investigate the developmental role of PKCepsilon in the prostaglandin F(2alpha)(PGF(2alpha))-induced rise in [Ca(2+)](i) and the induced decline in progesterone accumulation in cultures of cells isolated from the bovine corpus luteum. PGF(2alpha) increased [Ca(2+)](i) in Day 4 large luteal cells (LLCs), but the response was significantly lower than in Day 10 LLCs (4.3 +/- 0.6, n = 116 vs. 21.3 +/- 2.3, n = 110). Similarly, the fold increase in the PGF(2alpha)-induced rise in [Ca(2+)](i) in Day 4 small luteal cells (SLCs) was lower than in Day 10 SLCs (1.6 +/- 0.2, n = 198 vs. 2.7 +/- 0.1, n = 95). A PKCepsilon inhibitor reduced the PGF(2alpha)-elicited calcium responses in both Day 10 LLCs and SLCs to 3.5 +/- 0.3 (n = 217) and 1.3 +/- 0.1 (n = 205), respectively. PGF(2alpha) inhibited LH-stimulated progesterone (P(4)) accumulation only in the incubation medium of Day 10 luteal cells. Both conventional and PKCepsilon-specific inhibitors reversed the ability of PGF(2alpha) to decrease LH-stimulated P(4) accumulation, and the PKCepsilon inhibitor was more effective at this than the conventional PKC inhibitor. In conclusion, the evidence indicates that PKCepsilon, an isozyme expressed in corpora lutea with acquired PGF(2alpha) luteolytic capacity, has a regulatory role in the PGF(2alpha)-induced Ca(2+) signaling in luteal steroidogenic cells, and that this in turn may have consequences (at least in part) on the ability of PGF(2alpha) to inhibit LH-stimulated P(4) synthesis at this developmental stage.     

Multiple inhibitory effects of a luteinizing hormone-releasing hormone agonist on hCG-dependent steroidogenesis and on FSH-dependent responses in ovarian cells in vivo and in vitro

[125I] labelled [D-Leu6, des-Gly-NH10(2)] LH-RH ethylamide (LH-RHa), when injected into immature female rats, bound specifically not only to the pituitary but also to the ovaries. LH-RHa inhibited hCG-stimulated progesterone production and ovarian weight augmentation in hypophysectomized immature female rats in vivo. FSH-induced ovarian hCG receptors and ovarian weight gain in diethylstilbestrol (DES)-treated hypophysectomized immature female rats were also suppressed by LH-RHa. Progesterone production by rat luteal cells in vitro was inhibited by LH-RHa. LH-RHa did not change the affinity or population of LH/hCG receptor in porcine granulosa cells in short term incubation. However, LH-RHa inhibited induction of LH/hCG receptor stimulated by FSH and insulin in long term culture of porcine granulosa cells. LH-RHa delayed hCG-stimulated cyclic AMP accumulation in porcine granulosa cells. These findings suggest that LH-RHa inhibits hCG-stimulated cyclic AMP accumulation and subsequent progesterone production as well as FSH-stimulated LH/hCG receptor induction by acting directly on ovarian cells.     

Action of GnRH on steroid secretion by luteal cells of cyclic and early pregnant sows in vitro

The effect of GnRH was studied on progesterone (P4), oestradiol-17 beta (E2) and testosterone (T) secretion by porcine luteal cells from the 13th day of the oestrous cycle and the 18th day of pregnancy. Trypsin-dispersed luteal cells (5 X 10(4) cells/ml) were incubated in medium 199 with 10% calf serum with or without GnRH in doses of 0.1, 1, 10 and 100 mg/ml and with 1 microgram LH and 50 U/ml hCG. The concentration of P4, E2 and T in the medium was estimated by radioimmunological method after 6 hours of incubation. The results showed that GnRH had no effect on the secretion of the investigated steroid hormones by luteal cells from cyclic sows. GnRH at a dose of 10 g inhibited E2 secretion and at a dose of 1 ng T secretion by cells from pregnant sows. LH and hCG stimulated release of P4 by luteal cells in both physiological stages. The conclusion drawn was that GnRH does not act directly on luteal cells of cyclic sows but may inhibit E2 and T secretion by cells of pregnant sows.     

Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells.

The murine receptor for luteinizing hormone (LHR) was cloned and expressed in L cells. This LHR (mature protein of 674 amino acids) is very similar to that of the rat (same length, 36 amino acid differences) but differs significantly more from that of man (673 amino acids, 109 differences). Expression of the murine LHR in L cells led to the appearance of binding sites for human chorionic gonadotropin (hCG) with a Kd of 150 pM and an LH- and hCG-stimulable adenylyl cyclase activity (EC50 = 50-100 pM hCG). Upon labeling pools of phosphoinositides with [3H]myo-inositol, L cells expressing the murine LHR responded to hCG with an increase in their rate of phosphoinositide hydrolysis (EC50 = 2,400 pM hCG). This was accompanied by an increase in intracellular Ca2+ [( Ca2+]i), as determined by the Fura2 method. This increase in [Ca2+]i in response to hCG was dependent on the LHR, for HCG did not affect [Ca2+]i in L cells not expressing the LHR. The effect was not due to the cAMP-forming activity of the LH receptor, for neither forskolin nor prostaglandin E1, which both increase cAMP levels in L cells, had a similar effect in either control or LHR-expressing cells and isoproterenol had no effect in L cells expressing a functionally active hamster beta-adrenergic receptor. The effect was also not due to overexpression of a Gs-coupled receptor, for L cells expressing 8-fold higher levels of the human V2 vasopressin receptor did not mimic the Ca(2+)-mobilizing response of the LH receptor. We conclude that the LH receptor has the capability of activating two intracellular signaling pathways: one leading to stimulation of adenylyl cyclase and resulting in increases in cAMP and a second leading to stimulation of phospholipase C and resulting in formation of inositol phosphates and elevations in [Ca2+]i. These data correlate positively with and provide a mechanistic explanation for previous reports on the ability of hCG to mobilize phosphoinositides and increasing [Ca2+]i in luteal and granulosa cells (e.g. Davis, J. S., West, L. A., and Farese, R. V. (1984) J. Biol. Chem. 259, 15028-15034).     

The regulation of steroidogenesis by opioid peptides in porcine theca cells

The present study was designed to investigate basal and LH-induced steroidogenesis in porcine theca cells from large follicles in response to various concentrations (1-1000 nM) of mu opioid receptor agonists (beta-endorphin, DAMGO, FK 33-824), delta receptor agonists (met-enkephalin, leu-enkephalin, DPLPE) and kappa receptor agonists (dynorphin A, dynorphin B, U 50488). Agonists of mu opioid receptors suppressed basal androstenedione (A4), testosterone (T) and oestradiol-17beta (E2) secretion and enhanced LH-induced A4 and T release by theca cells. The inhibitory effect of the agonists on E2 secretion was abolished in the presence of LH. All delta receptor agonists depressed basal progesterone (P4) output. However, the influence of these agents on LH-treated cells was negligible. Among delta receptor agonist used only leu-enkephalin and DPLPE at the lowest concentrations inhibited basal A4 release. The presence of LH in culture media changed the influence of these opioids from inhibitory to stimulatory. Similarly, DPLPE reduced T secretion by non-stimulated theca cells and enhanced T secretion of stimulated cells. All of delta agonists inhibited basal E2 secretion and unaffected its release from LH-treated theca cells. Agonists of kappa receptors inhibited basal, non-stimulated, P4 secretion and two of them (dynorphin B, U 50488) potentiated LH-induced P4 output. Basal A4 and T release remained unaffected by kappa agonist treatment, but the cells cultured in the presence of LH generally increased both androgen production in response to these opioids. Basal secretion of E2 was also suppressed by kappa agonists. This inhibitory effect was not observed when the cells were additionally treated with LH. In view of these findings we suggest that opioid peptides derived from three major opioid precursors may directly participate in the regulation of porcine theca cell steroidogenesis.     

Evidence that basal secretion of relaxin by individual cultured large luteal cells is influenced by mobilization of intracellular calcium: analysis by a reverse hemolytic plaque assay.

Ca2+ redistribution from an intracellular site(s) is a key biochemical event associated with relaxin (RLX) secretion by large luteal cells (LLCs) of porcine origin. However, the functional significance of internal stores of Ca2+ to basal rates of RLX secretion is not well understood. In addition, the identity of the intracellular storage site(s) for Ca2+ within LLCs is not known, nor is it clear if all RLX-releasing LLCs are equally dependent on this pool. In the present study, release of RLX from 24 h cultured luteal cells derived from early pregnant swine was monitored by a reverse hemolytic plaque assay (RHPA). Incubation of cultures in the presence of graded concentrations of thapsigargin (1 nM-1 microM), a plant sesquiterpene lactone that inhibits endoplasmic reticulum Ca(2+)-ATPase and thereby increases cytosolic Ca2+ concentrations, resulted in a dose-related increase in basal RLX secretion. The stimulatory effect of thapsigargin on RLX production was not abrogated by culture in Ca(2+)-free medium. Suppression of Ca2+ release from the endoplasmic reticulum of LLCs, achieved by incubating monolayers in medium containing dantrolene (1-100 microM), resulted in dose-related inhibition of basal RLX release. Taken together, these results suggest that the endoplasmic reticulum serves as a major storage site for Ca2+ redistribution within LLCs and, furthermore, that mobilization from this site is functionally coupled to basal secretion of RLX.     

c-erbB_2对大鼠黄体细胞hCG诱导的孕酮分泌的影响

采用离体细胞体外孵育法 ,研究反义c erbB2 寡脱氧核苷酸 (antisensec erbB2 ODN)对大鼠黄体细胞hCG诱导的孕酮分泌的影响 ,及其与外源性cAMP和Ca2 以及蛋白抑制剂放线菌酮 (CYX)之间的关系。结果表明 ,反义c erbB2 以剂量相关方式抑制黄体细胞hCG诱导的孕酮的产生 ,同时使c erbB2 蛋白染色阳性的黄体细胞百分数下降 ,无义tatODN没有相应的作用。10 -4 mol/L的二丁酰cAMP能明显反转反义c erbB2 ODN对孕酮产生和c erbB2 表达的抑制作用 ,钙离子通道阻断剂维拉帕米和蛋白抑制剂CYX对此抑制作用有协同效应。该实验说明c erbB2 参于hCG诱导黄体细胞生孕酮作用     

Expression and localization of luteinizing hormone receptor in the female mouse reproductive tract

The presence of the LH receptor (LHR) in nongonadal tissues of the reproductive tract has been reported, but localization studies have not been performed. Our objectives were to demonstrate the presence of LHR in the reproductive tract and to localize receptor expression. Reproductive age rats and mice were obtained and (125)I-hCG binding assays were performed on membrane preparations from the uterus, ovary, liver, and testis. In situ hybridizations were performed using (35)S-labeled antisense and sense RNA probes prepared from nucleotides 1-591 of the mouse LHR cDNA. Specific hCG binding was detected in membrane preparations from the ovary, uterus, and testis but not in the liver in both the rat and mouse. In the ovary, LHR mRNA was localized in theca cells, large follicles, and corpora lutea as expected. In the uterus, LHR mRNA was expressed in stromal cells of the endometrium and in the uterine serosa. Uterine smooth muscle cells had low levels of expression, and the endometrial epithelium was negative. In the oviduct, high levels of LHR expression were noted on the serosa and in subepithelial cells. Oviductal smooth muscle had low expression, and the epithelium was negative. We conclude that functional, nongonadal LHR are expressed in the mouse reproductive tract. The presence and localization of LHR expression in the mouse reproductive tract lay the foundation for transgenic models to address the physiologic role of these receptors.     

Leutropin/beta-adrenergic receptor chimeras bind choriogonadotropin and adrenergic ligands but are not expressed at the cell surface.

In some G-protein-coupled receptors (e.g. beta-adrenergic receptor (beta 2 AR)), the ligand-binding pocket is contained within the hydrophobic transmembrane domain. In others (e.g. luteinizing hormone receptor (LHR)), the relative roles of the extracellular N-terminal domain and the transmembrane region in hormone binding are unknown. To study the roles of these domains, we prepared vectors encoding the rat LHR N-terminal domain alone (L- -), the LHR N-terminal domain fused to the transmembrane and C-terminal domains of the vesicular stomatitis virus-G protein (LVV), the LHR N-terminal domain fused to the transmembrane and C-terminal domains of the hamster beta 2 AR (LAA), and the beta 2 AR N-terminal domain fused to the transmembrane and C-terminal domains of the rat LHR (ALL). Membrane preparations obtained from COS-7 cells expressing the beta 2 AR or LAA bound the beta-adrenergic antagonist 125I-cyanopindolol with equal affinity, confirming the observation that the beta 2 AR transmembrane domain forms the hormone-binding site. Membranes from COS-7 cells transfected with LHR bound 125I-human choriomic gonadotropin (hCG). However, membranes from LAA-, L(- -)-, and LVV-transfected cells had low capacity to bind 125I-hCG unless they were solubilized with Triton X-100. The affinity of the detergent-solubilized receptors for 125I-hCG was similar to that of the LHR. We were unable to detect binding of 125I-hCG to ALL in the presence or absence of detergent. These observations suggest that, whereas the transmembrane region of the beta 2 AR is sufficient to bind adrenergic ligands, the N-terminal region of the LHR is required for binding of hCG. Although the N terminus of the LHR is sufficient to bind hCG, both the N terminus and the transmembrane domains of the LHR are required for receptor expression on the cell surface.     

The physiological role of beta-endorphin in porcine ovarian follicles

Beta-endorphin-like immunoreactivity (beta-END-LI) was measured by radioimmunoassay in porcine ovarian follicular fluid (FF) from small, medium and large follicles throughout the oestrous cycle. The concentration of beta-END-LI in FF from small follicles collected on days 1-5 of the cycle was at least tenfold higher than in the fluid from any other follicles independently from their size and the period of the cycle. The level of beta-END-LI in small follicles on days 6-10 was drastically decreased. Subsequently, on days 11-16 its concentration was enhanced and reduced again in pre-ovulatory period of the cycle. Concentrations of beta-END-LI in FF from medium follicles were relatively equal throughout the cycle (days 6-21). No significant differences in beta-END-LI levels were found between small, medium and large follicles from days 17-21. However, beta-END-LI concentrations in medium follicles on days 11-13 and 14-16 were statistically lower than those in small follicles. Moreover, the effects of FSH, prolactin (PRL), progesterone (P4), testosterone (T) and 17 beta-oestradiol (E2) on beta-END-LI release by granulosa cells (GCs) from large follicles and, on the other hand, the effects of the opioid agonist FK 33-824 alone or in combination with FSH, PRL or naloxone (NAL) on follicular steroidogenesis were studied. FSH drastically increased beta-END-LI output in a dose-dependent fashion. This stimulatory effect of the gonadotrophin was inhibited by the highest dose of P4 (10(-5) M). The effect of PRL and the steroids added to the cultures on beta-END-LI release was negligible. FSH- or PRL-induced P4 secretion by GCs was essentially abolished by both FK 33-824 and NAL. However, androstenedione (A4) and testosterone output by the cells was greatly potentiated by FK 33-824. In the presence of NAL, FSH or PRL, A4 release stimulated by FK 33-824 was suppressed to the basal level. Secretion of E2 was completely free from the influence of FK 33-824 or NAL; only oestrone (E1) output was modulated by them in cultures where FSH or PRL was present. In conclusion, FSH appears to be the key regulator of beta-END-LI secretion by porcine granulosa cells. Moreover, steroidogenesis in pig granulosa cells is modulated by opioid peptides acting both alone and by way of interaction with FSH or PRL.     

Assessment of luteal rescue and desensitization of macaque corpus luteum brought about by human chorionic gonadotrophin and deglycosylated human chorionic gonadotrophin treatment

The objective of the current study was to investigate the mechanism by which the corpus luteum (CL) of the monkey undergoes desensitization to luteinizing hormone following exposure to increasing concentration of human chorionic gonadotrophin (hCG) as it occurs in pregnancy. Female bonnet monkeys were injected (im) increasing doses of hCG or dghCG beginning from day 6 or 12 of the luteal phase for either 10 or 4 or 2 days. The day of oestrogen surge was considered as day ‘0’ of luteal phase. Luteal cells obtained from CL of these animals were incubated with hCG (2 and 200 pg/ml) or dbcAMP (2.5,25 and 100 M) for 3h at 37°C and progesterone secreted was estimated. Corpora lutea of normal cycling monkeys on day 10/16/22 of the luteal phase were used as controls. In addition thein vivo response to CG and deglycosylated hCG (dghCG) was assessed by determining serum steroid profiles following their administration. hCG (from 15–90 IU) but not dghCG (15-90 IU) treatment in vivo significantly (P < 0.05) elevated serum progesterone and oestradiol levels. Serum progesterone, however, could not be maintained at a elevated level by continuous treatment with hCG (from day 6–15), the progesterone level declining beyond day 13 of luteal phase. Administering low doses of hCG (15-90 IU/day) from day 6–9 or high doses (600 IU/day) on days 8 and 9 of the luteal phase resulted in significant increase (about 10-fold over corresponding control P < 0.005) in the ability of luteal cells to synthesize progesterone (incubated controls) in vitro. The luteal cells of the treated animals responded to dbcAMP (P < 0.05) but not to hCC added in vitro. The in vitro response of luteal cells to added hCG was inhibited by 0,50 and 100% if the animals were injected with low (15-90 IU) or medium (100 IU) between day 6–9 of luteal phase and high (600 IU on day 8 and 9 of luteal phase) doses of dghCG respectively; such treatment had no effect on responsivity of the cells to dbcAMP. The luteal cell responsiveness to dbcAMP in vitro was also blocked if hCG was administered for 10 days beginning day 6 of the luteal phase. Though short term hCG treatment during late luteal phase (from days 12—15) had no effect on luteal function, 10 day treatment beginning day 12 of luteal phase resulted in regain ofin vitro responsiveness to both hCG (P < 0.05) and dbcAMP (P < 0.05) suggesting that luteal rescue can occur even at this late stage. In conclusion, desensitization of the CL to hCG appears to be governed by the dose/period for which it is exposed to hCG/dghCG. That desensitization is due to receptor occupancy is brought out by the fact that (i) this can be achieved by giving a larger dose of hCG over a 2 day period instead of a lower dose of the hormone for a longer (4 to 10 days) period and (ii) the effect can largely be reproduced by using dghCG instead of hCG to block the receptor sites. It appears that to achieve desensitization to dbcAMP also it is necessary to expose the luteal cell to relatively high dose of hCG for more than 4 days     

Functional and structural regression of the rabbit corpus luteum is associated with altered luteal immune cell phenotypes and cytokine expression patterns

Following attenuation of progesterone production corpora lutea are selectively cleared, a process associated with recruitment of macrophages. In the rabbit little is known about luteal immune cell phenotypes and expression of cytokines, which influence immune cells and resident luteal cells, during luteolysis. Consequently, we studied luteal immune cells by immunohistochemistry as well as luteal IL-10, TNFalpha, MCP-1, IFN-gamma, and IL-1beta mRNA expression by semiquantitative RT-PCR from day 8 to day 20 in pseudopregnant rabbits (d8-d20 p.hCG). Luteal function was assayed by serum progesterone levels. Functional luteolysis commenced by d14 p.hCG as indicated by attenuation of serum progesterone levels. X4(+) tissue macrophage levels increased transiently on d12 and d14 p.hCG, whereas CD5(+) T-cell levels transiently declined on these two days. CD68(+) macrophages increased progressively after d16 p.hCG. The luteal mRNA level of the anti-inflammatory cytokine IL-10 as well as the mRNA levels of the pro-inflammatory cytokines TNFalpha and MCP-1 increased after d16 p.hCG and remained elevated up to d20 p.hCG. IFN-gamma and IL-1beta mRNA expression did not vary systematically. In summary, luteolysis was associated with an initial transient increase of X4(+) macrophages and decrease of CD5(+) T-cells, and later recruitment of CD68(+) macrophages. During structural regression pro- and anti-inflammatory cytokines are upregulated possibly to control immune cell function.     

Nuclear volume and chromatin conformation of small and large bovine luteal cells: effect of gonadotropins and prostaglandins and dependence on luteal phase

Summary Change in nuclear volume and chromatin conformation are generally considered to reflect altered gene expression in eukaryotic cells. The present studies were undertaken to investigate whether these nuclear parameters of luteal cells can be altered by hormone treatment in vitro or change during the estrous cycle. The nuclear volume of small luteal cells was significantly lower than that of large luteal cells during the cycle and pregnancy. The nuclear volumes of small and large luteal cells from pregnancy did not change during incubation without any hormone or with 10 nM prostaglandin (PG)F₂. However, incubation with 1 nM human chorionic gonadotropin (hCG) or 10 nM PGE₁ resulted in a significant increase of nuclear volume of small luteal cells by 4 h and that of large luteal cells by 6 h. Small cells were more responsive to hCG than large luteal cells. The nuclear volumes of small and large luteal cells also significantly increased from early to mid luteal phase with no further change in late luteal phase. hCG and PGE₁, as well as PGF₂, treatment resulted in a change of chromatin conformation of small and large luteal cells. Dibutyryl cyclic AMP (10 mM) mimicked the hormones by increasing nuclear volumes and changing the chromatin conformation of small and large luteal cells. Chromatin conformation of small and large luteal cells also changed from early to mid luteal phase and mid to late luteal phase. In conclusion, in vitro, hCG and PGs can regulate nuclear volume and/or chromatin conformation of small as well as large bovine luteal cells. In vivo, these nuclear changes occur during the periods of luteal growth, development and regression in the estrous cycle.