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Interplay between nitric oxide and gonadotrophin-releasing hormone in the neuromodulation of the corpus luteum during late pregnancy in the rat
Reproductive Biology and Endocrinology volume 20, Article number: 19 (2022)
Nitric oxide and GnRH are biological factors that participate in the regulation of reproductive functions. To our knowledge, there are no studies that link NO and GnRH in the sympathetic ganglia. Thus, the aim of the present work was to investigate the influence of NO on GnRH release from the coeliac ganglion and its effect on luteal regression at the end of pregnancy in the rat.
The ex vivo system composed by the coeliac ganglion, the superior ovarian nerve, and the ovary of rats on day 21 of pregnancy was incubated for 180 min with the addition, into the ganglionic compartment, of L-NG-nitro arginine methyl ester (L-NAME), a non-selective NO synthase inhibitor. The control group consisted in untreated organ systems.
The addition of L-NAME in the coeliac ganglion compartment decreased NO as well as GnRH release from the coeliac ganglion. In the ovarian compartment, and with respect to the control group, we observed a reduced release of GnRH, NO, and noradrenaline, but an increased production of progesterone, estradiol, and expression of their limiting biosynthetic enzymes, 3β-HSD and P450 aromatase, respectively. The inhibition of NO production by L-NAME in the coeliac ganglion compartment also reduced luteal apoptosis, lipid peroxidation, and nitrotyrosine, whereas it increased the total antioxidant capacity within the corpora lutea.
Collectively, the results indicate that NO production by the coeliac ganglion modulates the physiology of the ovary and luteal regression during late pregnancy in rats.
In the ovary, the corpus luteum (CL) is a transitory glandular structure, which is essential for the maintenance of pregnancy, given its ability to secrete progesterone. The function and lifespan of this dynamic gland depends on the balance between luteotrophic and luteolytic factors, such as steroid and peptide hormones, growth factors, gonadotropins, prostaglandins, cytokines, neuropeptides, and reactive oxygen species (ROS), among others . At the end of pregnancy in the rat, the CL undergoes a regression process characterized by decreased functionality and structural involution . Given the importance of this process for the homeostasis of the ovarian tissue, and to ensure the successful delivery of the fetuses, a series of autocrine and paracrine signals participate in its regulation.
Nitric oxide (NO) is a key cellular signaling factor in reproductive physiology. Several studies have demonstrated that NO participates in the control of gonadotrophin-releasing hormone (GnRH) production modulating the hypothalamo-pituitary-ovary axis. Chachlaki et al.  showed that the administration in vitro of a NO donor to hypothalamic explants stimulated GnRH release in a dose-dependent manner. Meanwhile, the local and in vivo inhibition of hypothalamic neural nitric oxide synthase (nNOS) activity led to disruption in the rat oestrus cyclicity. In addition to long-term regulation by the hypothalamic-pituitary axis, autonomic innervation represents a form of rapid and direct regulation of ovarian function that can be an important adaptation of female reproductive function to internal or external environmental changes . In rats, the main source of noradrenaline (NA) in the ovary is the superior ovarian nerve (SON); its fibers originate from the coeliac ganglion (CG) and are associated with follicular development and steroid secretion from the ovary [5, 6]. Recent findings support the view that these ganglia are complex integrative centers that receive inputs from central and peripheral sources with an abundance of neurochemical mediators, including NO . Specifically, the nNOS enzyme has been detected in fibers that innervate the CG [7, 8], and it has been reported that NO plays a role as a neurotransmitter and neuromodulator of synaptic transmission [9, 10]. In adition, the presence of the GnRH receptor was observed in coeliac and superior mesenteric ganglia of goats . Our group has already stardardized an integrated ex vivo system composed of the coeliac ganglion-superior ovarian nerve-ovary (CG-SON-Ovary), which mimics well the in vivo condition. This allows studying the interrelation of neural and endocrine phenomena on the physiology of the ovary. Using this model system, we reported that the CG is able to respond to different stimuli that modify the ovarian physiology through the SON in different reproductive states of the rat [10, 12,13,14,15]. Recently, we demonstrated the presence of GnRH/GnRH-receptor system in the CG and provided evidence for the fact that GnRH from the CG may trigger neuronal signals that promote luteal regression in late pregnant rats . However, to the best of our knowledge, there are no references about the relatioship between NO and GnRH affecting the function of the sympathetic ganglia. Herein, an ex-vivo CG-SON-Ovary model obtained from rats on day 21 of pregnancy was used to test whether the inhibition of NO synthesis in the CG compartment affects the release of GnRH from the CG, thus modulating the physiology of the ovary at the end of pregnancy.
Materials and methods
Virgin Holtzman strain female rats weighing 250 ± 50 g were used on day 21 of pregnancy. They were kept in the bioterium of the National University of San Luis (San Luis, Argentina) under rigorous light conditions (12 h light, 07:00–19:00, and 12 h darkness), controlled temperature (22 ± 2 °C), with water and food ad libitum. The procedure used to induce gestation was described previously in detail . Animals were handled according to the procedures approved in the UFAW Handbook on the Care and Management of Laboratory Animals. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the National University of San Luis (protocol number B-264/17).
Surgical and experimental procedures
The surgical procedure to remove the CG–SON–Ovary system and the incubation conditions were conducted according to Casais et al. . The animals were anaesthetized with 80 mg/kg of ketamine (Holliday Scott, Buenos Aires, Argentina) and 10 mg/kg of xylazine (Richmond, Buenos Aires, Argentina), intraperitoneally injected. The fetuses were removed and killed in an atmosphere of carbon dioxide. The CG–SON–Ovary system was extracted, and the mothers were sacrificed by decapitation. The entire CG–SON–Ovary system was removed by surgery, avoiding contact between the surgical instruments and the nerve fibers in order to prevent spontaneous nerve depolarization. The pieces of tissues removed consisted of the left ovary, the fibers that constituted the SON inserted in the suspensory ligament, and the CG accompanied by some small ganglia surrounding it. The entire surgical procedure was completed in 1–2 min. The CG–SON–Ovary system was placed in a cuvette with two isolated compartments, one for the CG and the other for the Ovary, both joined by the SON. The incubation medium used was Krebs-Ringer-bicarbonate buffer, with a pH of 7.4 with the addition of 0.1 mg/mL glucose and 0.1 mg/mL albumin at 37 °C, in an atmosphere composed of 95% O2 and 5% CO2. A schematic diagram of the experimental model was included in Fig.1. The ex vivo system was pre-incubated for 30 min, and the end of this period was considered incubation time 0. After this time, the buffer was replaced in both compartments, and 0.1 mg/mL ascorbic acid was added to the ganglion compartment as an antioxidant agent. The experimental group consisted in the addition in the ganglionic compartment of L-NG-nitro arginine methyl ester (L-NAME; Sigma-Aldrich, St. Louis, MO, USA), a non-selective NOS inhibitor, which was dissolved in 1 ml of Krebs-Ringer buffer at a final concentration of 100 μM . The control group consisted of CG–SON–Ovary systems that were untreated. The incubation was performed for 180 min. At the end of this period, the ganglionic and ovarian incubation liquids were extracted and kept at − 20 °C until the measurement of NO and GnRH. Also, NA, progesterone, and estradiol were measured in the ovarian incubation liquids. Whole ovaries were weighed, and the corpora lutea were separated and stored at − 80 °C until protein extraction. Whole ovaries were also fixed in Bouin liquid for further analysis by a terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay.
Levels of nitrites, water-soluble metabolites of NO, were measured spectrophotometrically in the ganglionic and ovarian incubation liquids. Samples were immediately mixed with Griess reagent (sulfanilamide with N-1-naphthyl-ethylendiamine–HCL). After a 10 min incubation period at room temperature, the optical readings at 540 nm were measured and the nmol of nitrite were determined using a standard curve. The assay sensitivity was < 2.5 nmol/mL. The intraassay coefficients of variation for all the assays were < 10.0%. The results were expressed as nmol of nitrite per milligram of ovarian tissue (nmol/mg ovary).
GnRH in ganglionic and ovarian incubation liquids was measured by RIA. Intra- and inter-assay coefficients of variation were 7.1 and 11.6%, respectively, and the standard curve maintained linearity up to 100 pg/tube of GnRH.
Progesterone and estradiol levels were measured in the ovarian incubation liquid using a RIA kit (Beckman Coulter and DIAsource, respectively, DiagnosMed SRL, Buenos Aires, Argentina) following the manufacturer’s instructions. The inter-and intra-assay coefficients of variation in all the assays were < 10.0%.
Levels of NA in ovarian incubation liquids were measured by high performance liquid chromatography (HPLC; Prominence, Shimadzu, Japan) with electrochemical detection (Coulochem III, ESA, MA, USA) at IACA Laboratorios (Buenos Aires, Argentina).
Enzyme-linked immunosorbent assay (ELISA)
Protein extracts were obtained using radioimmunoprecipitation assay buffer (RIPA buffer). The total protein concentration in corpora lutea homogenates was measured by the Bradford method.
The luteal expression of 3β-hydroxysteroid dehydrogenase (3β-HSD), an enzyme involved in progesterone synthesis, P450 aromatase (P450arom), involved in estradiol synthesis, and nitrotyrosine, a biomarker for the damage induced by NO-derived reactive nitrogen intermediates, were all analysed by enzyme-linked immunosorbent assay (ELISA). Aliquots containing 30 μg of total proteins for 3β-HSD, 20 μg of total proteins for P450arom, and 15 μg of total proteins for nitrotyrosine, were mixed with 0.1 M bicarbonate buffer with a pH of 9.6, in clear 96-well microplates (Corning Incorporated, Corning, NY, USA) and incubated for 1 h at 37 °C. After washing the sample with 0.05% (v/v) Tween 20 in phosphate buffered saline (PBS), and blocking it with 5% (w/v) non-fat dry milk in PBS with 0.05% (v/v) Tween 20 for 1 h at 37 °C, the microplates were incubated overnight at 4 °C with 100 μL of goat polyclonal anti-3β-HSD, 100 μL of rabbit polyclonal anti-P450arom (1:500; sc-30,820 and sc-30,086, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and 100 μL of rabbit polyclonal anti-nitrotyrosine antibody (1:1000; Sigma, St. Louis, MO, USA), respectively. After three washes, 100 μL of donkey anti-goat IgG peroxidase-linked antibody (1:500; sc-2020, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), 100 μL of goat anti-rabbit IgG–horse-radish peroxidase (HRP) conjugate (1:5000; sc-2004, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and 50 μL of goat anti-rabbit IgG-HRP conjugate (1:4000 dilution; Jackson Immuno Research Laboratories, West Grove, PA, USA) were added to each well and incubated for 1 h at 37 °C. Finally, immunocomplexes were quantified using 3,3′,5,5′-Tetramethylbenzidine (TMB). The oxidation reaction of the substrate was terminated with 2 M sulfuric acid, and the optical density at 450 nm was measured using a TECAN microplate reader (Infinite M200 PRO, Research Triangle Park, NC, USA). The results were expressed in arbitrary units.
The ovaries were processed for conventional optical microscopy; fixed in Bouin liquid, dehydrated in ethyl alcohol of increasing concentration, rinsed in xylol and included in paraffin. Four micrometer thick sections were generated with a Microm HM325 rotation microtome. Histological ovarian sections were processed for in situ localization of nuclei that exhibited DNA fragmentation using the In Situ Cell Death Detection Kit POD TUNEL assay (Cat N° 11,684,817,910 Roche, Basel, Switzerland), according to the manufacturer’s instructions. Apoptotic cells were visualized as immunolabelled intense brown structures after reaction with chromogen 3–3 ‘diaminobenzidine (DAB). Negative controls included omission of TdT. The positive control sections were incubated with 10 IU/mL DNase II (Sigma-Aldrich, St. Louis, MO, USA) in 50 mM Tris-HCL pH 7.5, 10 mM Mg2Cl, and 1 mg/mL BSA for 10 min at room temperature. Finally, the sections were counterstained with hematoxylin. The morphometric analysis of the histological sections processed with the TUNEL technique was performed using an Olympus BX40 optical microscope. Images were captured with a Sony SSC-DC5OA color camera and processed using the Image-Pro Plus 5.0 software. Three regularly spaced serial tissue sections (100 μm each) of corpora lutea corresponding to control and L-NAME groups were used and microscopic fields were examined under a 40X objective. In each section, 20 microscopic fields were randomly selected for each CL. The percentage of labeled cells was determined using the formula A/ (A + B) × 100, where A is the number of immunoreactive cells and B is the total number of unlabeled nuclei in the image. At least 1-hundred cells by field were counted by two independent observers, blinded to the experimental conditions. The results were expressed as a percentage of TUNEL positive cells, which was calculated per rat. All percentages were used to obtain the mean value per group.
Measurement of lipid peroxidation
The thiobarbituric acid reactive substances (TBARS) assay measures malondialdehyde (MDA) production from lipid hydroperoxides. A calibration curve was performed using 1,1,3,3-tetramethoxypropane as standard. TBARS were determined by the absorbance at 535 nm and were expressed as μmol of MDA per milligram of total proteins (μmol MDA/mg protein).
Total antioxidant capacity
Total antioxidant capacity (TAC) was measured by an improved method of bleaching of the 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS●+) by both lipophilic and hydrophilic antioxidants present in the protein extracts. The ABTS●+ was generated by oxidation of 7 mM ABTS with 2.45 mM potassium persulfate. The TAC was expressed as the percentage of reduction in the absorbance due to the ABTS●+, and it was determined as follows: % inhibition = [(A0-Af)/ A0] × 100, where A0 and Af are the absorbances at 734 nm of the reaction mixtures measured at t = 0 and after 5 min of sample addition, respectively. All measurements were performed in duplicate for each sample.
Statistical analysis was performed using GraphPad Prism (Version 5, GraphPad Software Inc. San Diego, CA, USA). All data are presented as the mean values ± standard error of the mean (S.E.M) in each group. Differences between two groups were analysed with Student’s unpaired t test. To analyze the percentage of positive TUNEL, a non-parametric (Mann–Whitney) test was used. A value of p < 0.05 was considered statistically significant. When comparing more than two groups, we used either one-way ANOVA followed by the Tukey’s multiple comparison test, or two-way ANOVA followed by the Bonferroni’s multiple comparison test.
Nitric oxide and GnRH ganglionic levels
In order to determine a possible role for NO in the release of GnRH in the CG, we first confirmed the NO release in the ganglionic compartment of CG-SON-Ovary organ complexes of control pregnant rats. Then, the addition of the NO synthesis blocker L-NAME to the ganglionic incubation medium effectively decreased the levels of nitrites (p < 0.01) (Fig. 2A), as well as GnRH (p < 0.05), compared to the control group (Fig. 2B), which suggests that NO may play a role in the release of GnRH in the CG.
Nitric oxide, GnRH and NA ovarian levels
NO, GnRH, and NA are neurotransmitters that participate in the modulation of ovarian physiology. The presence of L-NAME in the ganglion compartment significantly decreased the ovarian release of these neurotransmitters with respect to the control group (p < 0.05) (Fig. 3A-C).
Ovarian levels of progesterone and luteal expression of 3β-HSD
Given the previous findings by our group concerning the luteolytic effect of GnRH released from the CG on the pregnant rat ovary, we analysed the levels of the main luteal ovarian steroid—progesterone—and the luteal expression of its biosynthetic enzyme (3β-HSD). We observed that blocking NO synthesis in the CG compartment caused an increased release of progesterone in the ovarian compartment (p < 0.001) (Fig. 4A), while increased the expression of luteal 3β-HSD (p < 0.01) (Fig. 4B).
Ovarian levels of estradiol and luteal expression of P450arom
Estradiol is another key hormone regulating reproductive processes when acting on the CL. The addition of L-NAME in the ganglionic compartment increased the estradiol released in the ovarian compartment (P < 0.01) (Fig. 5A), as well as the expression of its limiting biosynthetic enzyme, P450arom, when compared to the control group (P < 0.05) (Fig. 5B).
After analyzing luteal steroidogenesis markers, we evaluated apoptosis as a marker of luteal structural regression. The corpora lutea isolated from the CG-SON-Ovary systems incubated with the addition of L-NAME in the ganglionic compartment showed a lower percentage of apoptotic cells in the ovarian compartment than the control group (p < 0.05) (Fig. 6).
We further analysed oxidative/nitrosative stress markers in the CL. The addition of L-NAME into the CG compartment significantly decreased the levels of TBARS (p < 0.05) (Fig. 7A), as well as nitrotyrosine (p < 0.01) (Fig. 7B). In addition, there was a significant increase in the TAC when compared to the control group (p < 0.05) (Fig. 7C).
GnRH and NO are signaling mediators that participate in the regulation of reproductive functions. Herein, by using an ex-vivo CG-SON-Ovary system we studied whether blocking NO synthesis in the CG affects the release of GnRH from the CG, and the impact on the physiology of the ovary at the end of pregnancy. Previous results from our research group demonstrated that the addition of the GnRH antagonist cetrorelix (CTX) to the ganglionic compartment of the CG-SON-Ovary system increased the ganglionic GnRH levels, indicating the presence of a functional GnRH system in the CG . Surprisingly, in that study we also found an increase in the levels of NO in the ganglionic compartment. Such increase could be a compensatory mechanism due to the blockage of the GnRH receptors and suggested a potential relationship between NO and GnRH release in the sympathetic ganglia.
Previous studies have suggested that NO is a key modulator of GnRH secretion from the hypothalamus, a necessary process for normal ovarian function and reproductive cyclicity [19, 20]. In sympathetic ganglia, the presence of the NO/NOS system has been established [7, 8]. Furthermore, the use of drugs that block NO synthesis, such as L-NAME, have shown that endogenously released NO can modulate synaptic transmission . In the present study, the addition of L-NAME decreased NO and GnRH levels in the ganglionic compartment. Therefore, these data suggest that NO affects the release of GnRH in the CG. Furthermore, we studied whether the joint blockage of the action of GnRH and the production of NO in the CG, through the combined addition of L-NAME and CTX in the ganglionic compartment, modifies the accumulation of GnRH. The results obtained indicate that the joint addition of L-NAME and CTX in the CG decreased the levels of GnRH in the ganglion (Supplementary Fig. 1A) and ovarian (Supplementary Fig. 1B) compartments compared to the control group. A comparative statistical analysis between the different experimental groups indicates the prevalence of the effect of L-NAME over CTX, reinforcing the role that NO plays in the accumulation of GnRH in the ganglionic compartment.
The inhibition of NO production in the CG compartment with L-NAME decreased the release of the gaseous neutrotransmitter in the ovarian compartment. This agrees with results showing a time-dependent decrease in nitrites levels upon 100 μM L-NAME treatment using the same multiorgan system in cycling rats (Supplementary Fig. 2). In addition to blocking the accumulation of GnRH in the ganglionic compartment, the addition of L-NAME in the CG compartment led to a decreased release of GnRH in the ovarian compartment. Taken together, these data suggest a link between NO and GnRH at the ganglionic and ovarian levels and, given the presence of GnRH and NO systems in the ovary, an intraovarian regulatory mechanism should not be ruled out.
Several studies have shown that GnRH may be an important regulator of the regression of the CL. Thus, the administration of GnRH agonists inhibits progesterone production and induces apoptosis in the CL of pregnant rats; such anti-steroidogenic effect may be due to the inhibitory effect on the enzymes involved in the steroidogenic pathway [21, 22]. In addition, an in vitro study showed a suppressive effect of a GnRH agonist on ovarian progesterone synthesis associated with decreased 3β-HSD in the ovary of mice . In agreement with this study, we demostrated that the increased progesterone biosynthesis in the CL was accompanied by an increase in the luteal expression of its limiting biosynthesis enzyme, 3β-HSD.
NA is a neurotransmitter that regulates ovarian steroidogenesis. Stimulatory or inhibitory effects of NA on steroid hormone secretion were reported, depending on what type of adrenoreceptors is activated in the ovary . The decrease in the release of NA from the ovary upon blocking NO synthesis in the CG indicates that, in addition to GnRH, NA may be one of the transmitters involved in the increase of progesterone production. In agreement with our results, Ramírez Hernández et al.  showed that sectioning of the SON reduces the levels of NA in the ovary, and that this effect is accompanied by an increase in the biosynthesis of progesterone. Regarding estradiol, Sridaran and Mahesh  demonstrated that GnRH has no effect on its luteal synthesis. However, it was demonstrated that the synthesis of estradiol is regulated negatively by NO in cells obtained from luteinized ovaries . Therefore, the decrease in ovarian NO production by L-NAME in the CG compartment may be responsible for the increase in estradiol biosynthesis and P450arom expression in the CL. These results were accompanied by a decrease in GnRH levels. This neuropeptide has not been detected in ovarian nerve endings so far. However, the presence of a GnRH system in the ovary is known, which is negatively regulated by estradiol [15, 28]. Therefore, in the present study, NO may regulate ovarian GnRH release through estradiol.
Apoptosis is one of the main mechanisms of celular death involved in the regression of the CL [2, 29]. Some reports indicate that the structural luteal regression begins before the functional luteal regression is complete [30, 31]. In addition, a modest number of apoptotic cells are present during the late luteal stage, which increases in the early postpartum period [32, 33]. Several studies support the protective role of progesterone in the function and survival of the CL, since it can locally stimulate its own production to protect the CL from cell death [2, 34, 35]. By contrast, GnRH may facilitate apoptosis and CL regression [16, 36, 37]. The reduced apoptotic levels found in the CL in the current study are consistent with an increase in the release of progesterone and a decrease in the release of GnRH into the medium of ovarian incubation of L-NAME-treated systems in the CG. In turn, the release of NA decreased in this experimental group. In addition, there is evidence that NA is involved in the regulation of ovarian apoptosis [15, 38]. Previous results from our laboratory indicate that on diestrus II, a stage in which the CL undergoes regression, the addition of NA in the ovarian incubation medium regulates ovarian steroidogenesis by modulating the release of GnRH, thus favoring apoptosis .
During luteal regression, the generation of ROS, which may be attributed to both accelerated generation and inefficient removal, has been associated with decreased progesterone production and induction of cell death [39,40,41]. In the present study, a decrease in the lipid peroxidation products and the nitrotyrosine concentration, together with low levels of NO, was observed in ovaries from systems incubated with L-NAME in the CG compartment. Regarding NO, both luteotrophic and luteolytic effects have been attributed to this molecule [42,43,44]. Furthermore, it has been suggested that the effect of NO on cellular processes depends on its concentration and the presence of other free radicals. Thus, the proapoptotic effect of NO is mainly associated with high concentrations [45, 46]. Therefore, the low levels of ovarian NO would contribute to the low percentage of apoptotic cells seen in the L-NAME group. Nitrotyrosine is indicative of formation and activity of the NO-derived oxidant peroxynitrite; therefore, it serves as a marker of NO metabolism ; it should be noted that its concentration also decreased in this experimental group.
Regarding lipid peroxidation, several studies indicate that its increase inhibits progesterone production in luteal cells and is a trigger for luteal regression [39, 48]. In the present study, in addition to the decrease in the levels of these oxidative/nitrosative damage markers, an increase in the TAC was found in the corpora lutea isolated from the CG-SON-Ovary systems incubated with L-NAME in the CG compartment. The ovarian tissue contains enzymatic and non-enzymatic antioxidant systems, responsible for eliminating the excess of ROS, which could be the subject of endocrine regulation [49, 50]. It should be noted that the aforementioned results occurred with an increase in estradiol levels, to which several studies have attributed antioxidant properties in the luteal cells [51, 52]. For example, Vega et al.  reported that the addition of estradiol to human luteal cells incubated in vitro led to a decrease in TBARS levels.
It is not yet well established which factors have the greatest contribution to the inhibition of progesterone synthesis, activation of the signaling pathway of apoptosis, and regression of the CL. However, it was suggested by other authors that the end of luteal functionality depends on the balance among several factors [1, 48]. In a previous study, we provided evidence that GnRH released from in the CG might trigger neuronal signals that promote luteal regression in late pregnancy . In the present study, we showed that NO affects GnRH release from the CG and the consequent impacts on the physiology of the ovary. Together, these findings highlight the functional role of these factors through the peripheral innervation in the regulation of ovarian function. Given that the modulation of the NO system can be a useful tool in the process of induction of labor, in addition to the known effect on the uterus, the contribution of the peripheral neural pathway and its influence on ovarian steroid hormone production should be taken into account.
The inhibition of NO production by L-NAME in the GC lead to a reduction in ganglionic GnRH release and caused changes that promoted luteal wellbeing, associated with a decrease in ovarian levels of NO, GnRH and NA, possible neurotransmitters involved in the observed effects. These observations suggest that the production of NO by the CG modulates the physiology of the ovary and the regression of the CL during late pregnancy in rats.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. This work is part of the CONICET Doctoral Scholarship and the Doctoral Thesis of Laura Morales.
Coeliac ganglion-superior ovarian nerve-ovary
L-NG-nitro arginine methyl ester
Reactive oxygen species
Neural nitric oxide synthase
Enzyme-linked immunosorbent assay
Thiobarbituric acid reactive substances
Terminal deoxynucleotidyl transferase dUTP nick-end labelling
Davis JS, Rueda BR. Recent advancements in corpus luteum development, function, maintenance and regression: Forum introduction. Reprod Biol Endocrinol. 2003;1:86.
Stocco C, Telleria C, Gibori G. The molecular control of corpus luteum formation, function, and regression. Endocr Rev. 2007;28:117–49.
Chachlaki K, Garthwaite J, Prevot V. The gentle art of saying NO: how nitric oxide gets things done in the hypothalamus. Nat Rev Endocrinol. 2017;13:521–35.
Uchida S, Kagitani F. Autonomic nervous regulation of ovarian function by noxious somatic afferent stimulation. J Physiol Sci. 2015;65:1–9.
Baljet B, Drukker J. The extrinsic innervation of the abdominal organs in the female rat. Acta Anat (Basel). 1979;104:243–67.
Lawrence IE Jr, Burden HW. The origin of the extrinsic adrenergic innervation to the rat ovary. Anat Rec. 1980;196:51–9.
Kaestner CL, Smith EH, Peirce SG, Hoover DB. Immunohistochemical analysis of the mouse celiac ganglion: an integrative relay station of the peripheral nervous system. J Comp Neurol. 2019;527:2742–60.
Domoto T, Teramoto M, Tanigawa K, Tamura K, Yasui Y. Origins of nerve fibers containing nitric oxide synthase in the rat celiac-superior mesenteric ganglion. Cell Tissue Res. 1995;281:215–21.
Quinson N, Catalin D, Miolan JP, Niel JP. Nerve-induced release of nitric oxide exerts dual effects on nicotinic transmission within the coeliac ganglion in the rabbit. Neuroscience. 1998;84:229–40.
Delsouc MB, Della Vedova MC, Ramírez D, Delgado SM, Casais M. The production of nitric oxide in the coeliac ganglion modulates the effect of cholinergic neurotransmission on the rat ovary during the preovulatory period. Nitric Oxide. 2018;75:85–94.
Fan J, Chen W, Guo X, Wang Z, Ming J, Guo Y, et al. Distribution of GnRH receptor in celiac-superior mesenteric ganglia of goat and its implications. Acta Vet Zootech Sin. 2013;44:289–94.
Casais M, Delgado SM, Sosa Z, Rastrilla AM. Pregnancy in rats is modulated by ganglionic cholinergic action. Reproduction. 2006;131:1151–8.
Vallcaneras SS, Delgado SM, Motta A, Telleria CM, Rastrilla AM, Casais M. Effect of prolactin acting on the coeliac ganglion via the superior ovarian nerve on ovarian function in the postpartum lactating and non-lactating rat. Gen Comp Endocrinol. 2013;184:1–8.
Delgado SM, Escudero CG, Casais M, Gordillo M, Anzulovich AC, Sosa Z, et al. Ovaric physiology in the first oestral cycle: influence of noradrenergic and cholinergic neural stimuli from coeliac ganglion. Steroids. 2010;75:685–94.
Bronzi CD, Orozco AS, Rodriguez D, Rastrilla AM, Sosa ZY, Casais M. Noradrenaline modulates the presence of gonadotropin-releasing hormone in ovary. The importance of its interrelation on the ovarian steroidogenesis and apoptosis on dioestrus II in rat. J Steroid Biochem Mol Biol. 2015;154:39–46.
Morales L, Vallcaneras S, Delsouc MB, Filippa V, Aguilera-Merlo C, Fernández M, et al. Neuromodulatory effect of GnRH from coeliac ganglion on luteal regression in the late pregnant rat. Cell Tissue Res. 2021;384:487–98.
Casais M, Sosa ZY, Rastrilla AM, Aguado L. Coeliac ganglion adrenergic activity modifies ovarian progesterone during pregnancy: its inter-relationship with LH. J Endocrinol. 2001;170:575–84.
Casais M, Delgado SM, Vallcaneras S, Sosa Z, Rastrilla AM. Nitric oxide in prepubertal rat ovary: contribution of the ganglionic nitric oxide synthase system via superior ovarian nerve. Neuro Endocrinol Lett. 2007;28:39–44.
McCann SM, Haens G, Mastronardi C, Walczewska A, Karanth S, Rettori V, et al. The role of nitric oxide (NO) in control of LHRH release that mediates gonadotropin release and sexual behavior. Curr Pharm Des. 2003;9:381–90.
Bellefontaine N, Hanchate NK, Parkash J, Campagne C, de Seranno S, Clasadonte J, et al. Nitric oxide as key mediator of neuron-to-neuron and endothelia-to-glia communication involved in the neuroendocrine control of reproduction. Neuroendocrinology. 2011;93:74–89.
Sridaran R, Philip GH, Li H, Culty M, Liu Z, Stocco DM, et al. GnRH agonist treatment decreases progesterone synthesis, luteal peripheral benzodiazepine receptor mRNA, ligand binding and steroidogenic acute regulatory protein expression during pregnancy. J Mol Endocrinol. 1999;22:45–54.
Papadopoulos V, Dharmarajan AM, Li H, Culty M, Lemay M, Sridaran R. Mitochondrial peripheral-type benzodiazepine receptor expression. Correlation with gonadotropin-releasing hormone (GnRH) agonist-induced apoptosis in the corpus luteum. Biochem Pharmacol. 1999;58:1389–93.
Singh P, Krishna A. Effects of GnRH agonist treatment on steroidogenesis and folliculogenesis in the ovary of cyclic mice. J Ovarian Res. 2010;3:26.
Weiss GK, Dail WG, Ratner A. Evidence for direct neural control of ovarian steroidogenesis in rats. J Reprod Fertil. 1982;65:507–11.
Ramírez Hernández DA, Vieyra Valdez E, Rosas Gavilán G, Linares Culebro R, Espinoza Moreno JA, Chaparro Ortega A, et al. Role of the superior ovarian nerve in the regulation of follicular development and steroidogenesis in the morning of diestrus 1. J Assist Reprod Genet. 2020;37:1477–88.
Sridaran R, Mahesh VB. Suppression of luteal estradiol receptors and progesterone synthesis by a gonadotropin-releasing hormone agonist (WY-40972) during midgestation. Biol Reprod. 1989;40:276–82.
Olson LM, Jones-Burton CM, Jablonka-Shariff A. Nitric oxide decreases estradiol synthesis of rat luteinized ovarian cells: possible role for nitric oxide in functional luteal regression. Endocrinology. 1996;137:3531–9.
Nathwani PS, Kang SK, Cheng KW, Choi KC, Leung PC. Regulation of gonadotropin-releasing hormone and its receptor gene expression by 17beta-estradiol in cultured human granulosa-luteal cells. Endocrinology. 2000;141(5):1754–63.
Tang Z, Huang Y, Zhang Z, Tang Y, Chen J, Sun F, et al. Accumulated autophagosomes and excessive apoptosis during the luteal development of pregnant rats. Int J Clin Exp Pathol. 2017;10:11384–92.
Gaytán F, Bellido C, Morales C, Sánchez-Criado JE. Cyclic changes in the responsiveness of regressing corpora lutea to the luteolytic effects of prolactin in rats. Reproduction. 2001;122:411–7.
González-Fernández R, Martínez-Galisteo E, Gaytán F, Bárcena JA, Sánchez-Criado JE. Changes in the proteome of functional and regressing corpus luteum during pregnancy and lactation in the rat. Biol Reprod. 2008;79:100–14.
Telleria CM, Goyeneche AA, Cavicchia JC, Stati AO, Deis RP. Apoptosis induced by antigestagen RU486 in rat corpus luteum of pregnancy. Endocrine. 2001;15:147–55.
Kurusu S, Suzuki K, Taniguchi K, Yonezawa T, Kawaminami M. Structural regression of the rat corpus luteum of pregnancy: relationship with functional regression, apoptotic cell death, and the suckling stimulus. Zool Sci. 2009;26:729–34.
Telleria CM, Stocco CO, Stati AO, Deis RP. Progesterone receptor is not required for progesterone action in the rat corpus luteum of pregnancy. Steroids. 1999;64:760–6.
Accialini P, Hernández SF, Bas D, Pazos MC, Irusta G, Abramovich D, et al. A link between notch and progesterone maintains the functionality of the rat corpus luteum. Reproduction. 2015;149:1–10.
Kawaminami M, Shibata Y, Yaji A, Kurusu S, Hashimoto I. Prolactin inhibits annexin 5 expression and apoptosis in the corpus luteum of pseudopregnant rats: involvement of local gonadotropin-releasing hormone. Endocrinology. 2003;144:3625–31.
Endo T, Kiya T, Kitajima Y, Honnma H, Chida M, Hayashi T, et al. Identical changes in Bax expression, but not Fas ligand expression, occur in structural luteolysis in gonadotropin releasing hormone agonist- and prolactin-treated superovulated rats. Life Sci. 2005;76:2159–69.
Zhang L, Gao J, Cui S. miR-21 is involved in norepinephrine-mediated rat granulosa cell apoptosis by targeting SMAD7. J Mol Endocrinol. 2017;58:199–210.
Riley JC, Behrman HR. In vivo generation of hydrogen peroxide in the rat corpus luteum during luteolysis. Endocrinology. 1991;128:1749–53.
Nagaosa K, Shiratsuchi A, Nakanishi Y. Concomitant induction of apoptosis and expression of monocyte chemoattractant protein-1 in cultured rat luteal cells by nuclear factor-kappaB and oxidative stress. Develop Growth Differ. 2003;45:351–9.
Tanaka M, Miyazaki T, Tanigaki S, Kasai K, Minegishi K, Miyakoshi K, et al. Participation of reactive oxygen species in PGF2alpha-induced apoptosis in rat luteal cells. J Reprod Fertil. 2000;120:239–45.
Motta AB, Estevez A, de Gimeno MF. The involvement of nitric oxide in corpus luteum regression in the rat: feedback mechanism between prostaglandin F(2alpha) and nitric oxide. Mol Hum Reprod. 1999;5:1011–6.
Dong YL, Gangula PR, Fang L, Yallampalli C. Nitric oxide reverses prostaglandin-induced inhibition in ovarian progesterone secretion in rats. Hum Reprod. 1999;14:27–32.
Hurwitz A, Finci-Yeheskel Z, Milwidsky A, Mayer M. Regulation of cyclooxygenase activity and progesterone production in the rat corpus luteum by inducible nitric oxide synthase. Reproduction. 2002;123:663–9.
Thomas DD, Ridnour LA, Isenberg JS, Flores-Santana W, Switzer CH, Donzelli S, et al. The chemical biology of nitric oxide: implications in cellular signaling. Free Radic Biol Med. 2008;45:18–31.
Keator CS, Schreiber DT, Hoagland TA, McCracken JA. Luteotrophic and luteolytic effects of nitric oxide in sheep are dose-dependent in vivo. Domest Anim Endocrinol. 2008;35:74–80.
Stamler JS, Singel DJ, Loscalso J. Biochemistry of nitric oxide and its redox-activated forms. Science. 1992;258:1898–902.
Al-Gubory KH, Garrel C, Faure P, Sugino N. Roles of antioxidant enzymes in corpus luteum rescue from reactive oxygen species-induced oxidative stress. Reprod BioMed Online. 2012;25:551–60.
Aten RF, Kolodecik TR, Rossi MJ, Debusscher C, Behrman HR. Prostaglandin f2alpha treatment in vivo, but not in vitro, stimulates protein kinase C-activated superoxide production by nonsteroidogenic cells of the rat corpus luteum. Biol Reprod. 1998;59:1069–76.
Foyouzi N, Cai Z, Sugimoto Y, Stocco C. Changes in the expression of steroidogenic and antioxidant genes in the mouse corpus luteum during luteolysis. Biol Reprod. 2005;72:1134–41.
Murdoch WJ. Inhibition by oestradiol of oxidative stress-induced apoptosis in pig ovarian tissues. J Reprod Fertil. 1998;114:127–30.
Miyaguchi C, Muranaka S, Kanno T, Fujita H, Akiyama J, Yoshioka T, et al. 17beta-estradiol suppresses ROS-induced apoptosis of CHO cells through inhibition of lipid peroxidation-coupled membrane permeability transition. Physiol Chem Phys Med NMR. 2004;36:21–35.
Vega M, Castillo T, Retamales I, Las Heras J, Devoto L, Videla LA. Steroidogenic capacity and oxidative stress-related parameters in human luteal cell regression. Free Radic Biol Med. 1994;17:493–9.
This work was supported by Grant PROICO 2–2916-UNSL (to MC), and Fund 172858, Department of Pathology, McGill University (to CT).
Ethics approval and consent to participate
Animals were handled according to the procedures approved in the UFAW Handbook on the Care and Management of Laboratory Animals. The experimental protocol was approved by the University of San Luis Animal Care and Use Committee (number protocol: B-264/17).
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The authors declare that they have no competing interests.
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Effect of the combined addition of L-NAME (100 μM) and CTX (10− 6 M) in CG on the release of GnRH, at 180 min. Incubation of the CG-SON-Ovary system of rats with 21 days of pregnancy. A) Ganglionic GnRH release, expressed in pg/mg CG. B) Ovarian GnRH release, expressed in pg/mg Ovary. The Control group consisted of untreated CG-SON-Ovary systems. Values represent the mean ± S.E.M. of 6 animals per experimental group. One-way ANOVA followed by the Tukey’s multiple comparison tests: (*) P < 0.05; (●) P < 0.01; (■) P < 0.001.
Effect of stimulation of the CG with 100 μM L-NAME on the concentration of nitrites in the incubation medium of the left ovary, during the first proestrus in the rat. For this, the CG-SON-Ovary system was extracted from 37-day-old Holtzman rats and immediately placed in a cuvette with two compartments, one for the CG and the other for the ovary, both joined by the SON. The incubation medium was 1 ml of Krebs-Ringer bicarbonate buffer, pH 7.4, with 0.1 mg/ml dextrose and 0.1 mg/ml BSA at 37 °C in a saturated atmosphere of 95% O2 and 5% CO2. The results show that the addition of this nitric oxide synthase inhibitor in the ganglion compartment decreases the nitrite concentration in the ovarian incubation medium, throughout 180 min of incubation. The values represent the mean ± SEM of 6 animals per experimental group. Two-way ANOVA followed by Bonferroni’s multiple comparison tests: (*) P < 0.05; (■) P < 0.001.
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Vallcaneras, S., Morales, L., Delsouc, M.B. et al. Interplay between nitric oxide and gonadotrophin-releasing hormone in the neuromodulation of the corpus luteum during late pregnancy in the rat. Reprod Biol Endocrinol 20, 19 (2022). https://doi.org/10.1186/s12958-022-00894-6
- Nitric oxide
- Gonadotrophin-releasing hormone
- Ex-vivo coeliac ganglion-superior ovarian nerve-ovary system
- End of pregnancy
- Corpus luteum