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Luteinizing hormone receptors are expressed in rat myenteric neurons and mediate neuronal loss
Autonomic Neuroscience: Basic and Clinical 193 (2015) 104–107
Contents lists available at ScienceDirect
Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu
Luteinizing hormone receptors are expressed in rat myenteric neurons and mediate neuronal loss Elin Sand a, Ulrikke Voss a, Bodil Ohlsson b, Eva Ekblad a,⁎ a b
Department of Experimental Medical Science, Unit Neurogastroenterology, BMC B11, Lund University, Sölvegatan 19, SE 22184 Lund, Sweden Department of Clinical Sciences, Division of Internal Medicine, Lund University, Sweden Lund University, Inga Marie Nilssons gata 32, SE 21428 Malmö, Sweden
a r t i c l e
i n f o
Article history: Received 22 June 2015 Received in revised form 9 September 2015 Accepted 7 October 2015 Keywords: Enteric nervous system (ENS) Gonadotropin-releasing hormone (GnRH) Luteinizing hormone Primary cultures of enteric neurons
a b s t r a c t Background: Clinical observations have suggested repeated gonadotropin-releasing hormone (GnRH) exposure to cause intestinal dysfunction and loss of enteric neurons. This has been further studied and confirmed in a rat in vivo model involving iterated GnRH treatments. Mechanisms behind are enigmatic since no GnRH receptors are found to be expressed in enteric neurons neither in man nor rat. Both species, however, harbor substantial subpopulations of luteinizing hormone (LH) receptor-immunoreactive myenteric neurons which suggests that intestinal GnRH-induced neuropathy may be mediated by LH release. Aims: To reveal if exposures of GnRH or LH to rat myenteric neurons in vitro cause neuronal loss. Methods: Primary cultured adult rat myenteric neurons were exposed to single or repeated treatments of the GnRH analog buserelin or the LH analog lutrotropin alpha, and neuronal survival was determined by cell counting. Possible presence of GnRH- or LH receptor -immunoreactive neurons was determined by immunocytochemistry. Results: Exposure to the LH, but not the GnRH, analog caused significantly reduced neuronal survival. LH, but not GnRH, receptors were found to be expressed on cultured myenteric neurons. Conclusion: Myenteric neurons express LH receptors in vitro and LH exposure causes reduced neuronal survival. This suggests that GnRH-induced enteric neuropathy in vivo is mediated by way of LH release and activation of enteric neuronal LH receptors. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Gastrointestinal (GI) dysfunction with enteric neuropathy and neurodegeneration are often identified in patients diagnosed with e.g. enteric dysmotility (ED), chronic intestinal pseudo-obstruction (CIPO) and diabetes (Gabbard and Lacy, 2013; Knowles et al., 2013; Yarandi and Srinivasan, 2014). Gonadotropin releasing hormone (GnRH) stimulates secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) through GnRH receptor activation (Roch et al., 2014). Based on the observations that functional dysmotility is more common in women and that symptoms worsen during post luteal phase, pregnancies and when on oral contraceptives, GnRH analogs were put forward as a candidate for treating severe GI symptoms (Mathias et al., 1998). Women with functional GI disorders were evaluated in a double-blind placebo-controlled study involving continuous treatment with the GnRH analog leuprolide (Mathias et al., 1998). The analog reduced symptom scores for nausea and abdominal pain, and improved GI motility was found. Doubts on the use of GnRH agonists for treating ⁎ Corresponding author. E-mail addresses: [email protected] (E. Sand), [email protected] (U. Voss), [email protected] (B. Ohlsson), [email protected] (E. Ekblad).
http://dx.doi.org/10.1016/j.autneu.2015.10.001 1566-0702/© 2015 Elsevier B.V. All rights reserved.
IBS were, however, raised (Farthing, 1998). Such disbeliefs were reinforced when a woman undergoing multiple in vitro fertilization (IVF) with GnRH analogs was diagnosed with chronic intestinal pseudo obstruction (Ohlsson et al., 2007). Histological examination of the small intestine showed a reduced number of myenteric neurons. Additional cases of abdominal dysfunction after GnRH analog treatment were to follow (Hammar et al., 2013). Investigations into the underlying mechanisms, using an animal model mimicking IVF procedures, showed administration of the GnRH analog buserelin to rats to cause a significant loss of enteric neurons all along the GI tract after multiple (3–4) exposure periods (Sand et al., 2013a). Further T-lymphocyte infiltration in myenteric ganglia (Ohlsson et al., 2014) and reduced fecal weight with increased amount of fecal fat were identified (Sand et al., 2014). Throughout the GI tract in both man and rat no GnRH receptor expression is found (Sand et al., 2013b). Therefore, the marked loss of enteric neurons in response to GnRH administration is most likely indirectly mediated; possibly through LH or FSH release. To date no FSH receptor expression has been identified in the GI tract of neither man nor rat, while LH receptors are expressed in myenteric neurons in both (Sand et al., 2013b). This study aim to investigate if GnRH analog-induced enteric neuronal death, noted in vivo, is mediated by direct or indirect mechanisms,
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using in vitro cultures of primary rat myenteric neurons. Enteric neuronal survival was examined after single or repeated exposures of GnRH analog (buserelin) or recombinant human LH (lutropin alfa). In addition, the possible presence of GnRH and/or LH receptors on cultured myenteric neurons was investigated. 2. Methods 2.1. Animals Twenty female Sprague–Dawley rats (140–160 g, Charles River, DE) and two male Sprague–Dawley rats (300 g, Charles River, DE) were used. The rats were allowed to acclimatize to the climate- and lightcontrolled animal facility for at least 5 days before sacrifice. Standard rat chow (4% fat/g; Lactamin R36, SE) and water were supplied at all times. All experimental procedures were approved by the animal ethics committee in Lund and Malmö, Sweden. The animals were used in accordance with European Communities Council Directive (2010/63/EU) and the Swedish Animal Welfare Act (SFS 1988:534). The female rats were used for primary culturing of myenteric neurons while tissues from male rats were used for antibody evaluation. 2.2. Myenteric neuronal cultures Primary cultures of myenteric neurons were performed using a previously described method (Voss and Ekblad, 2014). In brief anaesthetized rats had their small intestine exposed. The longitudinal muscle with attached myenteric ganglia were stripped, without penetrating the gut mucosa. The rats were killed by heart puncture after tissue sampling. The tissue was cut into pieces (2 × 2 mm), washed in Ca2 + and Mg2 + free Hank's balanced salt solution (HBSS, Life Technologies, SE), treated with collagenase II (1.5 mg/ml, Life Technologies SE) and protease (1.5 mg/ml, Sigma-Aldrich, SE), for 25 min at 37 °C. Trypsin (1.25 mg/ml, BioChrom, DE) and EDTA (0.01%, ethylenediaminetetraacid, Sigma-Aldrich, SE) were added and incubated 20 min. Trypsin inhibition was by addition of 50% fetal calf serum (FCS, Life Technologies, SE). The cell suspension was centrifuged and washed in HBSS three times. The pellet was diluted in 2.6 ml Neurobasal A (NBA) culture medium containing 10% FCS, 0.5 mM L-glutamine, 50 U penicillin G sodium and 50 μg streptomycin sulphate per ml (Life Technologies, SE). Cultures were prepared by seeding 50 μl of the constantly mixed cell suspension into 8-well chambers (BD Falcon, VWR, SE) prefilled with 450 μl NBA culture media and incubated in a humidified incubator holding 5% CO2. From each animal six 8-well chambers (each well 69 mm2) were prepared. Cultures were never prepared by pooling cell suspensions from different rats. After 4 days in vitro (4 DIV), experimentation was initiated. 2.3. Experimental setup All agents were diluted in NBA cell culture medium; a flow chart of the experimental design is illustrated in Fig. 1. For all experiments untreated controls receiving NBA culture medium, were run in parallel. Single exposure experiments were performed by adding buserelin (10−10 – 10− 6M Suprefact®, Sanofi-Aventis, SE) or lutropin alfa (10−10 – 10−7M, Luveris®, MerckSerono, SE) to cultures followed by a 4 day incubation (DIV 4 + 4). The repeated exposure experiments were performed by adding buserelin (10−8 – 10−6 M) or lutropin alfa (10−10 – 10− 8 M) to cultures every other day for eight days (DIV 4 + 8). On day 5, 7, 9, and 11 cultures were exposed to pharmacological agent, on day 6, 8, 10, and 12 cultures were exposed to NBA culture medium. The possible expression of GnRH- and LH receptors on cultured myenteric neurons studied on six separate cultures, cultured for 8 days in NBA only (DIV 4 + 4), medium was changed on the 4th day.
Fig. 1. Schematic illustration of the experimental setup. Single exposure was by the addition of either the GnRH analog buserelin (10−10 – 10−6 M) or the LH analog lutropin alpha (10−10 – 10−7M) (grey boxes) to cultures after a 4 day incubation in NBA medium (white boxes) post seeding. Exposure time was 4 days followed by fixation. Repeated exposure started after a 4 day incubation in NBA medium only and was by the addition of buserelin (10−8 – 10−6M) or lutropin alpha (10−10 – 10−8 M) on days 5, 7, 9 and 11. In between the cultures were grown in NBA medium only.
All cultures were fixed in Stefanini's fixative for 30 min and rinsed in Tyrode's solution 2 × 15 min. In order to enhance antibody penetration, the cultures were frozen in −20 °C for at least 1 h before being processed for immunocytochemistry. 2.4. Immunocytochemistry Antibodies raised against the human neuronal marker (HuC/D) were used as a pan-neuronal marker. The cultures were rinsed in phosphate buffer containing 0.25% Triton X-100 (PBS-T), incubated with primary antibodies against HuC/D (dilution 1:400, code A-21271, produced in mouse; Life Technologies, SE; (Lin et al., 2003)) over night at 4 °C. For visualization, cultures were washed in PBS-T and exposed to DyLight TM 488-conjugated goat anti-mouse IgG antiserum (dilution 1:1000; Jackson Immunoresearch Laboratories, USA) for 1 h. Analysis on possible neuronal GnRH- or LH receptor expressions was by double immunolabeling using antibodies against HuC/D (dilution 1:400) and GnRH-receptor (dilution 1:800, produced in goat, code no Sc-8682, Santa Cruz Biotech Inc., USA; (Sand et al., 2013b)) or HuC/D (dilution 1:400) and LH receptor (dilution 1:1600, produced in rabbit, code no L6792, Sigma Aldrich; (Sand et al., 2013b)). For visualization, cultures were exposed to DyLight TM 594-conjugated donkey anti-mouse IgG in combination with either DyLight TM 488conjugated donkey anti-goat IgG (for GnRH receptor visualization) or DyLight TM 594-conjugated donkey anti-rabbit IgG (for LH receptor visualization) for 1 h (all 1:1000; Jackson Immunoresearch Laboratories, USA). Mounting was in PBS:glycerol 1:1 followed by fluorescence microscopy using appropriate filter settings. 2.5. Controls used in the immunocytochemical procedures Absorption control was performed using the primary antiserum against GnRH receptors inactivated by the addition of 100 μg of synthetic peptide (code no Sc-8682P, Santa Cruz Biotech Inc. USA) per mL diluted antiserum. Controls did not exhibit any immunostaining. Synthetic antigens for testing specificities of the HuC/D and the LH-receptor antibodies are not commercially available, why omission of the primary antibodies was used as control. For further characterization of GnRH- and LH receptor antisera, positive control tissues from rat were used. Pituitary and testis from two naïve male rats were fixed in Stefanini's overnight, rinsed three times in Tyrode's solution containing 10% sucrose, mounted in Tissue-Tek (Histolab, SE), frozen on dry ice, and sectioned (10 μm). Cryo sections from the pituitary were exposed to the GnRH receptor
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antiserum and intense immunoreactivity was observed in pituicytes. Cryo sections from testis were exposed to the LH receptor antiserum and intense immunoreactivity was observed in Leydig cells. 2.6. Cell counting Neuronal survival of cultured myenteric neurons was evaluated by counting the number of all HuC/D-immunoreactive (IR) neurons within each well. Neuronal survival was calculated as survival of untreated controls run in parallel. The percentages of neurons IR to GnRH- or LH receptors were evaluated by counting the number of Hu-IR neurons also IR for GnRHreceptors or LH receptors. At least 200 HuC/D-IR neurons were counted in each well. 2.7. Statistical analysis Results are presented as medians and spreads as 25th and 75th percentile or mean ± SEM. Statistical analyses were performed by the Mann–Whitney U test or the Kruskal–Wallis test followed by Dunn's multiple comparison tests. Differences were considered statistically significant when p b 0.05. n (number of culture wells) = 4–15. 3. Results 3.1. Presence of GnRH- and LH receptors on cultured myenteric neurons. Cultured myenteric neurons from rat small intestine displayed intense HuC/D immunoreactivity and were found single or in small clusters. The number of myenteric neurons/mm2 was 5.9 ± 0.5 (n = 21). Fibroblasts, glia and smooth muscle cells were also present within the cultures. No GnRH receptor-immunoreactive (IR) cells, including HuC/D-IR neurons, were identified within the cultures. Of cultured myenteric neurons 18.5 ± 1.1% (n = 14), were IR to LH receptors (Fig. 2). None of the non-neuronal cells within the cultures were LH receptor-IR. 3.2. Effects of single or multiple GnRH or LH exposures on enteric neuronal survival. Single dose exposure to buserelin (10−10 – 10−6) or lutropin alfa (10−10 – 10− 7 M) for 4 days did not change neuronal survival compared to untreated controls run in parallel (Fig. 3, left panel). Repeated exposures, as described in Methods and in Fig. 1, to the GnRH analog buserelin (10− 8 – 10−6 M) did not affect neuronal survival (Fig. 3, right panel). However repeated exposures of lutropin alfa (10−10 – 10−8 M) significantly reduced neuronal survival to approximately 50% (10−10 M = p b 0.05; 10−9and 10−8 M = p b 0.01) compared to controls run in parallel (Fig. 3, right panel).
4. Discussion The main findings presented in this study are that LH receptors are expressed by about 19% of cultured rat myenteric neurons and that repeated exposure of LH induces neuronal loss in vitro. The LH receptor belongs to the rhodopsin/β2 adrenergic receptor-like family A of G protein coupled receptor, predominantly expressed in the gonads (Menon and Menon, 2012). The receptor binds both LH and human chorionic gonadotropin (HCG) with high affinity and has been found to be expressed extra gonadal e.g. in brain, urinary bladder, retina (Rao and Lei, 2007), pancreatic beta-cells (Parkash et al., 2015) and enteric neurons (Sand et al., 2013a, 2013b). Chronic stimulation of GnRH analogs initially increases the secretion of LH and FSH from the pituitary. However, after about 10 days of treatment, desensitization occurs and gonadotropin secretion ceases (Belchetz et al., 1978). Thus GnRH agonists are useful in several different clinical settings, e.g. sex-hormone-dependent neoplasms, endometriosis, polycystic ovarian syndrome and IVF treatment. We have previously reported that repeated in vivo administration of the GnRH analog buserelin causes a significant reduction (50%) of myenteric neurons in fundus, ileum and colon (Sand et al., 2013a). We further showed that, GnRH- and FSH receptors are absent throughout the GI tract of both man and rat. On the other hand, LH receptor IR was found in considerable subpopulations of submucous as well as myenteric neurons in both species (Sand et al., 2013a, 2013b). The relative number of neurons expressing LH receptors was further found to be markedly reduced after GnRH exposure in vivo, suggesting them to be particularly targeted (Sand et al., 2013a). These findings strongly suggest that repeated GnRH exposure leads to enteric neuronal loss through a mechanism involving LH receptor activation. Present findings that repeated, but not single, exposure of recombinant human LH to cultured adult rat myenteric neurons reduces survival and that 19% of cultured myenteric neurons are LH receptor-IR, support our theory of a direct LH receptormediated effect. As no GnRH receptors are present on enteric neurons and no GnRH-induced LH secretion can take place in vitro, the presence of buserelin is without effect on survival of cultured neurons. The mechanisms behind the enteric neuronal cell loss can only be speculated on. For comparison, LH stimulation of granulosa cells showed a dramatic change in gene transcripts coding for steroidogenic enzymes, cytoskeletal proteins and several signaling molecules coding for pro- and anti-apoptotic processes, as well as signaling molecules including GTP, cAMP and various kinases and phosphatases (Sasson et al., 2004). Most of the effects evoked by LH receptor stimulation seem to be mediated through cAMP/protein kinase A (PKA) (Sasson et al., 2004). LH receptor stimulation on human ovarian granulosa cells causes release of several bioactive mediators, e.g. estradiol, progesterone, pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP), and amphiregulin (Morelli et al., 2008; Breckwoldt et al., 1996). Both PACAP and VIP influence apoptosis and caspase-3 activation (Morelli et al., 2008). Further, HCG stimulation of granulosa cells increases cAMP concentration and induces extensive
Fig. 2. Rat ileum myenteric neurons cultured for 4 + 4 days and double immunostained for a) the general neuronal marker HuC/D (green) and b) luteinizing hormone (LH) receptors (red). Myenteric neurons are randomly spread throughout the culture dish and some of them are LH receptor-IR. In c) micrographs a) and b) are merged to illustrate the presence of myenteric neurons co-stained for both HuC/D and LH receptor-IR material (arrows). Arrowheads indicate LH receptor-IR nerve terminals. Bar represents 50 μm.
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Fig. 3. Left panel. Neuronal survival of cultured rat myenteric neurons treated with a single dose of buserelin (10−10 – 10−6 M) or lutropin alfa (10−10 – 10−7 M) for 4 days. No change in neuronal survival was noted compared to controls run in parallel. Right panel. Neuronal survival of rat myenteric neurons cultured in the presence of buserelin (10−8 – 10−6 M) or lutropin alfa (10−10 – 10−8 M) on days 5, 7, 9 and 11 in vitro (DIV) and in NBA only on 6, 8, 10 and 12 DIV. Repeated treatment with buserelin did not affect neuronal survival compared to controls run in parallel while repeated treatments with lutropin alfa (10−10 – 10−8 M) reduced neuronal survival to 43–52% compared to controls run in parallel. Results are presented as medians and spreads expressed as 25th and 75th percentile, n = 4–14. Statistical analysis was performed by Kruskal–Wallis test followed by Dunn's multiple comparison tests, differences were considered statistically significant when p b 0.05 and indicated by * = p b 0.05, ** = p b 0.01.
apoptosis (Breckwoldt et al., 1996). For comparison it is notable that a down-regulation of LH receptors is accompanied by decreased apoptosis (Srivastava and Krishna, 2011). We have previously reported that repeated GnRH exposure in vivo causes an increased relative number of activated caspase-3-IR enteric neurons prior to the neuronal loss (Sand et al., 2013a), indicative of apoptosis as the mechanism behind. The physiological role of enteric neuronal LH-receptors is unknown. In rat, LH/HCG has been reported to alter myoelectric activity in rat small intestine (Ducker et al., 1996), to inhibit gastric emptying (Seow et al., 2013) and to stimulate secretion of potassium and bicarbonate from pancreas and Brunner glands (Panesar and Poon, 1998). In man high levels of HCG have been ascribed an important cause of the cardinal symptoms, nausea and vomiting, in hyperemesis gravidarum (Tamay and Kuʂҫu, 2011). The enteric neuropathy occurring after iterated LH treatment in vitro (present study) and GnRH analog treatment (by way of LH release) in vivo (Sand et al., 2013a) indicate that exogenously administered reproductive hormone analogs may have serious and negative effects on the enteric nervous system. Present data strongly suggests a modulating role of LH (and HCG) on enteric neuronal activity and maintenance. Conflict of interest None. Ethical approval All applicable international, national and/or institutional guidelines for the care and use of animals were followed. Acknowledgements Grant support from the Faculty of Medicine, Lund, Påhlssons Foundation (2014/2793) and the Royal Physiographic Society in Lund (2014/2433). The funders took no role in study design, data collection and analysis, or in preparation of the manuscript. References Belchetz, P.E., Plant, T.M., Nakai, Y., et al., 1978. Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202, 631–633. Breckwoldt, M., Selvaraj, N., Aharoni, D., et al., 1996. Expression of Ad4-BP/cytochrome P450 side chain cleavage enzyme and induction of cell death in long-term cultures of human granulosa cells. Mol. Hum. Reprod. 2 (6), 391–400. Ducker, T.E., Boss, J.W., Altug, S.A., et al., 1996. Luteinizing hormone and human chorionic gonadotropin fragment the migrating myoelectric complex in rat small intestine. Neurogastroenterol. Motil. 8, 95–100.
Farthing, M.J.G., 1998. New drugs in the management of the irritable bowel syndrome. Drugs 56, 11–21. Gabbard, S.L., Lacy, B.E., 2013. Chronic intestinal pseudo-obstruction. Nutr. Clin. Pract. 3, 307–316. Hammar, O., Roth, B., Bengtsson, M., et al., 2013. Autoantibodies and gastrointestinal symptoms in infertile women in relation to in vitro fertilization. BMC Pregnancy Childbirth 13, 201. http://dx.doi.org/10.1186/1471-2393-13-201. Knowles, C.H., Lindberg, G., Panza, E., et al., 2013. New perspectives in the diagnosis and management of enteric neuropathies. Nat. Rev. Gastroenterol. Hepatol. 4, 206–218. Lin, Z., Sandgren, K., Ekblad, E., 2003. Increased expression of vasoactive intestinal polypeptide in cultured myenteric neurons from adult rat small intestine. Auton. Neurosci. 107, 9–19. Mathias, J.R., Clench, M.H., Abell, T.L., et al., 1998. Effect of leuprolide acetate in treatment of abdominal pain and nausea in premenopausal women with functional bowel disease. A double-blind, placebo-controlled, randomized study. Dig. Dis. Sci. 43, 1347–1355. Menon, K.M., Menon, B., 2012. Structure, function and regulation of gonadotropin receptors—a perspective. Mol. Cell. Endocrinol. 356 (1–2), 88–97. Morelli, M.B., Barberi, M., Gambardella, A., et al., 2008. Characterization, expression and functional activity of pituitary adenylate cyclase-activating polypeptide and its receptors in human granulosa-luteal cells. J. Clin. Endocrinol. Metab. 93 (12), 4924–4932. Ohlsson, B., Veress, B., Janciauskiene, S., et al., 2007. Chronic intestinal pseudo-obstruction due to buserelin-induced formation of anti-GnRH antibodies. Gastroenterology 132, 45–51. Ohlsson, B., Sand, E., Veress, B., 2014. Ganglioneuritis is common in rats with enteric neuropathy due to buserelin treatment. Regul. Pept. 190–191, 43–45. Panesar, N.S., Poon, C.W., 1998. HCG: its pancreatic and duodenal receptors and in vivo electrolyte secretion in female rats. Am. J. Physiol. 275, G1430–G1436. Parkash, J., Lei, Z., Rao, C.V., 2015. The presence of human chorionic gonadotropin/luteinizing hormone receptors in pancreatic β-cells. Reprod. Sci. http://dx.doi.org/10.1177/ 1933719115570910. Rao, C.V., Lei, Z.M., 2007. The past, present and future of nongonadal LH/hCG actions in reproductive biology and medicine. Mol. Cell. Endocrinol. 269 (1–2), 2–8. Roch, G.J., Busby, E.R., Sherwood, N.M., 2014. GnRH receptors and peptides: skating backward. Gen. Comp. Endocrinol. 209, 118–134. Sand, E., Voss, U., Hammar, O., et al., 2013a. Gonadotropin-releasing hormone analog buserelin causes neuronal loss in rat gastrointestinal tract. Cell Tissue Res. 351, 521–534. Sand, E., Bergvall, M., Ekblad, E., et al., 2013b. Expression and distribution of GnRH, LH, and FSH and their receptors in gastrointestinal tract of man and rat. Regul. Pept. 187, 24–28. Sand, E., Roth, B., Weström, B., et al., 2014. Structural and functional consequences of buserelin-induced enteric neuropathy in rat. BMC Gastroenterol. 14, 209. http://dx. doi.org/10.1186/s12876-014-0209-7. Sasson, R., Rimon, E., Dantes, A., et al., 2004. Gonadotrphin-induced gene regulation in human granulosa cells obtained from IVF patients. Modulation of steroidogenic genes, cytoskeletal genes and genes coding for apoptotic signalling and protein kinases. Mol. Hum. Reprod. 10 (5), 299–311. Seow, K.-M., Lee, J.-L., Doong, M.-L., et al., 2013. Human chorionic gonadotropin regulates gastric emptying in ovariectomized rats. J. Endocrinol. 216, 307–314. Srivastava, R.K., Krishna, A., 2011. Increased circulating leptin level inhibits folliculogenesis in vespertilionid bat, Scotophilus Heathii. Mol. Cell. Endocrinol. 337 (1–2), 24–35. Tamay, A.G., Kuʂҫu, N.K., 2011. Hyperemesis gravidarum: current aspects. J. Obstet. Gynaecol. 31, 708–712. Voss, U., Ekblad, E., 2014. Lipopolysaccharide-induced loss of cultured rat myenteric neurons—role of AMP-activated protein kinase. PLoS One 9 (12), e114044. http:// dx.doi.org/10.1371/journal.pone.0114044. Yarandi, S.S., Srinivasan, S., 2014. Diabetic gastrointestinal motility disorders and the role of enteric nervous system: current status and future directions. Neurogastroenterol. Motil. 5, 611–624.
Contents lists available at ScienceDirect
Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu
Luteinizing hormone receptors are expressed in rat myenteric neurons and mediate neuronal loss Elin Sand a, Ulrikke Voss a, Bodil Ohlsson b, Eva Ekblad a,⁎ a b
Department of Experimental Medical Science, Unit Neurogastroenterology, BMC B11, Lund University, Sölvegatan 19, SE 22184 Lund, Sweden Department of Clinical Sciences, Division of Internal Medicine, Lund University, Sweden Lund University, Inga Marie Nilssons gata 32, SE 21428 Malmö, Sweden
a r t i c l e
i n f o
Article history: Received 22 June 2015 Received in revised form 9 September 2015 Accepted 7 October 2015 Keywords: Enteric nervous system (ENS) Gonadotropin-releasing hormone (GnRH) Luteinizing hormone Primary cultures of enteric neurons
a b s t r a c t Background: Clinical observations have suggested repeated gonadotropin-releasing hormone (GnRH) exposure to cause intestinal dysfunction and loss of enteric neurons. This has been further studied and confirmed in a rat in vivo model involving iterated GnRH treatments. Mechanisms behind are enigmatic since no GnRH receptors are found to be expressed in enteric neurons neither in man nor rat. Both species, however, harbor substantial subpopulations of luteinizing hormone (LH) receptor-immunoreactive myenteric neurons which suggests that intestinal GnRH-induced neuropathy may be mediated by LH release. Aims: To reveal if exposures of GnRH or LH to rat myenteric neurons in vitro cause neuronal loss. Methods: Primary cultured adult rat myenteric neurons were exposed to single or repeated treatments of the GnRH analog buserelin or the LH analog lutrotropin alpha, and neuronal survival was determined by cell counting. Possible presence of GnRH- or LH receptor -immunoreactive neurons was determined by immunocytochemistry. Results: Exposure to the LH, but not the GnRH, analog caused significantly reduced neuronal survival. LH, but not GnRH, receptors were found to be expressed on cultured myenteric neurons. Conclusion: Myenteric neurons express LH receptors in vitro and LH exposure causes reduced neuronal survival. This suggests that GnRH-induced enteric neuropathy in vivo is mediated by way of LH release and activation of enteric neuronal LH receptors. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Gastrointestinal (GI) dysfunction with enteric neuropathy and neurodegeneration are often identified in patients diagnosed with e.g. enteric dysmotility (ED), chronic intestinal pseudo-obstruction (CIPO) and diabetes (Gabbard and Lacy, 2013; Knowles et al., 2013; Yarandi and Srinivasan, 2014). Gonadotropin releasing hormone (GnRH) stimulates secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) through GnRH receptor activation (Roch et al., 2014). Based on the observations that functional dysmotility is more common in women and that symptoms worsen during post luteal phase, pregnancies and when on oral contraceptives, GnRH analogs were put forward as a candidate for treating severe GI symptoms (Mathias et al., 1998). Women with functional GI disorders were evaluated in a double-blind placebo-controlled study involving continuous treatment with the GnRH analog leuprolide (Mathias et al., 1998). The analog reduced symptom scores for nausea and abdominal pain, and improved GI motility was found. Doubts on the use of GnRH agonists for treating ⁎ Corresponding author. E-mail addresses: [email protected] (E. Sand), [email protected] (U. Voss), [email protected] (B. Ohlsson), [email protected] (E. Ekblad).
http://dx.doi.org/10.1016/j.autneu.2015.10.001 1566-0702/© 2015 Elsevier B.V. All rights reserved.
IBS were, however, raised (Farthing, 1998). Such disbeliefs were reinforced when a woman undergoing multiple in vitro fertilization (IVF) with GnRH analogs was diagnosed with chronic intestinal pseudo obstruction (Ohlsson et al., 2007). Histological examination of the small intestine showed a reduced number of myenteric neurons. Additional cases of abdominal dysfunction after GnRH analog treatment were to follow (Hammar et al., 2013). Investigations into the underlying mechanisms, using an animal model mimicking IVF procedures, showed administration of the GnRH analog buserelin to rats to cause a significant loss of enteric neurons all along the GI tract after multiple (3–4) exposure periods (Sand et al., 2013a). Further T-lymphocyte infiltration in myenteric ganglia (Ohlsson et al., 2014) and reduced fecal weight with increased amount of fecal fat were identified (Sand et al., 2014). Throughout the GI tract in both man and rat no GnRH receptor expression is found (Sand et al., 2013b). Therefore, the marked loss of enteric neurons in response to GnRH administration is most likely indirectly mediated; possibly through LH or FSH release. To date no FSH receptor expression has been identified in the GI tract of neither man nor rat, while LH receptors are expressed in myenteric neurons in both (Sand et al., 2013b). This study aim to investigate if GnRH analog-induced enteric neuronal death, noted in vivo, is mediated by direct or indirect mechanisms,
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using in vitro cultures of primary rat myenteric neurons. Enteric neuronal survival was examined after single or repeated exposures of GnRH analog (buserelin) or recombinant human LH (lutropin alfa). In addition, the possible presence of GnRH and/or LH receptors on cultured myenteric neurons was investigated. 2. Methods 2.1. Animals Twenty female Sprague–Dawley rats (140–160 g, Charles River, DE) and two male Sprague–Dawley rats (300 g, Charles River, DE) were used. The rats were allowed to acclimatize to the climate- and lightcontrolled animal facility for at least 5 days before sacrifice. Standard rat chow (4% fat/g; Lactamin R36, SE) and water were supplied at all times. All experimental procedures were approved by the animal ethics committee in Lund and Malmö, Sweden. The animals were used in accordance with European Communities Council Directive (2010/63/EU) and the Swedish Animal Welfare Act (SFS 1988:534). The female rats were used for primary culturing of myenteric neurons while tissues from male rats were used for antibody evaluation. 2.2. Myenteric neuronal cultures Primary cultures of myenteric neurons were performed using a previously described method (Voss and Ekblad, 2014). In brief anaesthetized rats had their small intestine exposed. The longitudinal muscle with attached myenteric ganglia were stripped, without penetrating the gut mucosa. The rats were killed by heart puncture after tissue sampling. The tissue was cut into pieces (2 × 2 mm), washed in Ca2 + and Mg2 + free Hank's balanced salt solution (HBSS, Life Technologies, SE), treated with collagenase II (1.5 mg/ml, Life Technologies SE) and protease (1.5 mg/ml, Sigma-Aldrich, SE), for 25 min at 37 °C. Trypsin (1.25 mg/ml, BioChrom, DE) and EDTA (0.01%, ethylenediaminetetraacid, Sigma-Aldrich, SE) were added and incubated 20 min. Trypsin inhibition was by addition of 50% fetal calf serum (FCS, Life Technologies, SE). The cell suspension was centrifuged and washed in HBSS three times. The pellet was diluted in 2.6 ml Neurobasal A (NBA) culture medium containing 10% FCS, 0.5 mM L-glutamine, 50 U penicillin G sodium and 50 μg streptomycin sulphate per ml (Life Technologies, SE). Cultures were prepared by seeding 50 μl of the constantly mixed cell suspension into 8-well chambers (BD Falcon, VWR, SE) prefilled with 450 μl NBA culture media and incubated in a humidified incubator holding 5% CO2. From each animal six 8-well chambers (each well 69 mm2) were prepared. Cultures were never prepared by pooling cell suspensions from different rats. After 4 days in vitro (4 DIV), experimentation was initiated. 2.3. Experimental setup All agents were diluted in NBA cell culture medium; a flow chart of the experimental design is illustrated in Fig. 1. For all experiments untreated controls receiving NBA culture medium, were run in parallel. Single exposure experiments were performed by adding buserelin (10−10 – 10− 6M Suprefact®, Sanofi-Aventis, SE) or lutropin alfa (10−10 – 10−7M, Luveris®, MerckSerono, SE) to cultures followed by a 4 day incubation (DIV 4 + 4). The repeated exposure experiments were performed by adding buserelin (10−8 – 10−6 M) or lutropin alfa (10−10 – 10− 8 M) to cultures every other day for eight days (DIV 4 + 8). On day 5, 7, 9, and 11 cultures were exposed to pharmacological agent, on day 6, 8, 10, and 12 cultures were exposed to NBA culture medium. The possible expression of GnRH- and LH receptors on cultured myenteric neurons studied on six separate cultures, cultured for 8 days in NBA only (DIV 4 + 4), medium was changed on the 4th day.
Fig. 1. Schematic illustration of the experimental setup. Single exposure was by the addition of either the GnRH analog buserelin (10−10 – 10−6 M) or the LH analog lutropin alpha (10−10 – 10−7M) (grey boxes) to cultures after a 4 day incubation in NBA medium (white boxes) post seeding. Exposure time was 4 days followed by fixation. Repeated exposure started after a 4 day incubation in NBA medium only and was by the addition of buserelin (10−8 – 10−6M) or lutropin alpha (10−10 – 10−8 M) on days 5, 7, 9 and 11. In between the cultures were grown in NBA medium only.
All cultures were fixed in Stefanini's fixative for 30 min and rinsed in Tyrode's solution 2 × 15 min. In order to enhance antibody penetration, the cultures were frozen in −20 °C for at least 1 h before being processed for immunocytochemistry. 2.4. Immunocytochemistry Antibodies raised against the human neuronal marker (HuC/D) were used as a pan-neuronal marker. The cultures were rinsed in phosphate buffer containing 0.25% Triton X-100 (PBS-T), incubated with primary antibodies against HuC/D (dilution 1:400, code A-21271, produced in mouse; Life Technologies, SE; (Lin et al., 2003)) over night at 4 °C. For visualization, cultures were washed in PBS-T and exposed to DyLight TM 488-conjugated goat anti-mouse IgG antiserum (dilution 1:1000; Jackson Immunoresearch Laboratories, USA) for 1 h. Analysis on possible neuronal GnRH- or LH receptor expressions was by double immunolabeling using antibodies against HuC/D (dilution 1:400) and GnRH-receptor (dilution 1:800, produced in goat, code no Sc-8682, Santa Cruz Biotech Inc., USA; (Sand et al., 2013b)) or HuC/D (dilution 1:400) and LH receptor (dilution 1:1600, produced in rabbit, code no L6792, Sigma Aldrich; (Sand et al., 2013b)). For visualization, cultures were exposed to DyLight TM 594-conjugated donkey anti-mouse IgG in combination with either DyLight TM 488conjugated donkey anti-goat IgG (for GnRH receptor visualization) or DyLight TM 594-conjugated donkey anti-rabbit IgG (for LH receptor visualization) for 1 h (all 1:1000; Jackson Immunoresearch Laboratories, USA). Mounting was in PBS:glycerol 1:1 followed by fluorescence microscopy using appropriate filter settings. 2.5. Controls used in the immunocytochemical procedures Absorption control was performed using the primary antiserum against GnRH receptors inactivated by the addition of 100 μg of synthetic peptide (code no Sc-8682P, Santa Cruz Biotech Inc. USA) per mL diluted antiserum. Controls did not exhibit any immunostaining. Synthetic antigens for testing specificities of the HuC/D and the LH-receptor antibodies are not commercially available, why omission of the primary antibodies was used as control. For further characterization of GnRH- and LH receptor antisera, positive control tissues from rat were used. Pituitary and testis from two naïve male rats were fixed in Stefanini's overnight, rinsed three times in Tyrode's solution containing 10% sucrose, mounted in Tissue-Tek (Histolab, SE), frozen on dry ice, and sectioned (10 μm). Cryo sections from the pituitary were exposed to the GnRH receptor
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antiserum and intense immunoreactivity was observed in pituicytes. Cryo sections from testis were exposed to the LH receptor antiserum and intense immunoreactivity was observed in Leydig cells. 2.6. Cell counting Neuronal survival of cultured myenteric neurons was evaluated by counting the number of all HuC/D-immunoreactive (IR) neurons within each well. Neuronal survival was calculated as survival of untreated controls run in parallel. The percentages of neurons IR to GnRH- or LH receptors were evaluated by counting the number of Hu-IR neurons also IR for GnRHreceptors or LH receptors. At least 200 HuC/D-IR neurons were counted in each well. 2.7. Statistical analysis Results are presented as medians and spreads as 25th and 75th percentile or mean ± SEM. Statistical analyses were performed by the Mann–Whitney U test or the Kruskal–Wallis test followed by Dunn's multiple comparison tests. Differences were considered statistically significant when p b 0.05. n (number of culture wells) = 4–15. 3. Results 3.1. Presence of GnRH- and LH receptors on cultured myenteric neurons. Cultured myenteric neurons from rat small intestine displayed intense HuC/D immunoreactivity and were found single or in small clusters. The number of myenteric neurons/mm2 was 5.9 ± 0.5 (n = 21). Fibroblasts, glia and smooth muscle cells were also present within the cultures. No GnRH receptor-immunoreactive (IR) cells, including HuC/D-IR neurons, were identified within the cultures. Of cultured myenteric neurons 18.5 ± 1.1% (n = 14), were IR to LH receptors (Fig. 2). None of the non-neuronal cells within the cultures were LH receptor-IR. 3.2. Effects of single or multiple GnRH or LH exposures on enteric neuronal survival. Single dose exposure to buserelin (10−10 – 10−6) or lutropin alfa (10−10 – 10− 7 M) for 4 days did not change neuronal survival compared to untreated controls run in parallel (Fig. 3, left panel). Repeated exposures, as described in Methods and in Fig. 1, to the GnRH analog buserelin (10− 8 – 10−6 M) did not affect neuronal survival (Fig. 3, right panel). However repeated exposures of lutropin alfa (10−10 – 10−8 M) significantly reduced neuronal survival to approximately 50% (10−10 M = p b 0.05; 10−9and 10−8 M = p b 0.01) compared to controls run in parallel (Fig. 3, right panel).
4. Discussion The main findings presented in this study are that LH receptors are expressed by about 19% of cultured rat myenteric neurons and that repeated exposure of LH induces neuronal loss in vitro. The LH receptor belongs to the rhodopsin/β2 adrenergic receptor-like family A of G protein coupled receptor, predominantly expressed in the gonads (Menon and Menon, 2012). The receptor binds both LH and human chorionic gonadotropin (HCG) with high affinity and has been found to be expressed extra gonadal e.g. in brain, urinary bladder, retina (Rao and Lei, 2007), pancreatic beta-cells (Parkash et al., 2015) and enteric neurons (Sand et al., 2013a, 2013b). Chronic stimulation of GnRH analogs initially increases the secretion of LH and FSH from the pituitary. However, after about 10 days of treatment, desensitization occurs and gonadotropin secretion ceases (Belchetz et al., 1978). Thus GnRH agonists are useful in several different clinical settings, e.g. sex-hormone-dependent neoplasms, endometriosis, polycystic ovarian syndrome and IVF treatment. We have previously reported that repeated in vivo administration of the GnRH analog buserelin causes a significant reduction (50%) of myenteric neurons in fundus, ileum and colon (Sand et al., 2013a). We further showed that, GnRH- and FSH receptors are absent throughout the GI tract of both man and rat. On the other hand, LH receptor IR was found in considerable subpopulations of submucous as well as myenteric neurons in both species (Sand et al., 2013a, 2013b). The relative number of neurons expressing LH receptors was further found to be markedly reduced after GnRH exposure in vivo, suggesting them to be particularly targeted (Sand et al., 2013a). These findings strongly suggest that repeated GnRH exposure leads to enteric neuronal loss through a mechanism involving LH receptor activation. Present findings that repeated, but not single, exposure of recombinant human LH to cultured adult rat myenteric neurons reduces survival and that 19% of cultured myenteric neurons are LH receptor-IR, support our theory of a direct LH receptormediated effect. As no GnRH receptors are present on enteric neurons and no GnRH-induced LH secretion can take place in vitro, the presence of buserelin is without effect on survival of cultured neurons. The mechanisms behind the enteric neuronal cell loss can only be speculated on. For comparison, LH stimulation of granulosa cells showed a dramatic change in gene transcripts coding for steroidogenic enzymes, cytoskeletal proteins and several signaling molecules coding for pro- and anti-apoptotic processes, as well as signaling molecules including GTP, cAMP and various kinases and phosphatases (Sasson et al., 2004). Most of the effects evoked by LH receptor stimulation seem to be mediated through cAMP/protein kinase A (PKA) (Sasson et al., 2004). LH receptor stimulation on human ovarian granulosa cells causes release of several bioactive mediators, e.g. estradiol, progesterone, pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP), and amphiregulin (Morelli et al., 2008; Breckwoldt et al., 1996). Both PACAP and VIP influence apoptosis and caspase-3 activation (Morelli et al., 2008). Further, HCG stimulation of granulosa cells increases cAMP concentration and induces extensive
Fig. 2. Rat ileum myenteric neurons cultured for 4 + 4 days and double immunostained for a) the general neuronal marker HuC/D (green) and b) luteinizing hormone (LH) receptors (red). Myenteric neurons are randomly spread throughout the culture dish and some of them are LH receptor-IR. In c) micrographs a) and b) are merged to illustrate the presence of myenteric neurons co-stained for both HuC/D and LH receptor-IR material (arrows). Arrowheads indicate LH receptor-IR nerve terminals. Bar represents 50 μm.
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Fig. 3. Left panel. Neuronal survival of cultured rat myenteric neurons treated with a single dose of buserelin (10−10 – 10−6 M) or lutropin alfa (10−10 – 10−7 M) for 4 days. No change in neuronal survival was noted compared to controls run in parallel. Right panel. Neuronal survival of rat myenteric neurons cultured in the presence of buserelin (10−8 – 10−6 M) or lutropin alfa (10−10 – 10−8 M) on days 5, 7, 9 and 11 in vitro (DIV) and in NBA only on 6, 8, 10 and 12 DIV. Repeated treatment with buserelin did not affect neuronal survival compared to controls run in parallel while repeated treatments with lutropin alfa (10−10 – 10−8 M) reduced neuronal survival to 43–52% compared to controls run in parallel. Results are presented as medians and spreads expressed as 25th and 75th percentile, n = 4–14. Statistical analysis was performed by Kruskal–Wallis test followed by Dunn's multiple comparison tests, differences were considered statistically significant when p b 0.05 and indicated by * = p b 0.05, ** = p b 0.01.
apoptosis (Breckwoldt et al., 1996). For comparison it is notable that a down-regulation of LH receptors is accompanied by decreased apoptosis (Srivastava and Krishna, 2011). We have previously reported that repeated GnRH exposure in vivo causes an increased relative number of activated caspase-3-IR enteric neurons prior to the neuronal loss (Sand et al., 2013a), indicative of apoptosis as the mechanism behind. The physiological role of enteric neuronal LH-receptors is unknown. In rat, LH/HCG has been reported to alter myoelectric activity in rat small intestine (Ducker et al., 1996), to inhibit gastric emptying (Seow et al., 2013) and to stimulate secretion of potassium and bicarbonate from pancreas and Brunner glands (Panesar and Poon, 1998). In man high levels of HCG have been ascribed an important cause of the cardinal symptoms, nausea and vomiting, in hyperemesis gravidarum (Tamay and Kuʂҫu, 2011). The enteric neuropathy occurring after iterated LH treatment in vitro (present study) and GnRH analog treatment (by way of LH release) in vivo (Sand et al., 2013a) indicate that exogenously administered reproductive hormone analogs may have serious and negative effects on the enteric nervous system. Present data strongly suggests a modulating role of LH (and HCG) on enteric neuronal activity and maintenance. Conflict of interest None. Ethical approval All applicable international, national and/or institutional guidelines for the care and use of animals were followed. Acknowledgements Grant support from the Faculty of Medicine, Lund, Påhlssons Foundation (2014/2793) and the Royal Physiographic Society in Lund (2014/2433). The funders took no role in study design, data collection and analysis, or in preparation of the manuscript. References Belchetz, P.E., Plant, T.M., Nakai, Y., et al., 1978. Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202, 631–633. Breckwoldt, M., Selvaraj, N., Aharoni, D., et al., 1996. Expression of Ad4-BP/cytochrome P450 side chain cleavage enzyme and induction of cell death in long-term cultures of human granulosa cells. Mol. Hum. Reprod. 2 (6), 391–400. Ducker, T.E., Boss, J.W., Altug, S.A., et al., 1996. Luteinizing hormone and human chorionic gonadotropin fragment the migrating myoelectric complex in rat small intestine. Neurogastroenterol. Motil. 8, 95–100.
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