The present study demonstrates that AEA effectively reverses the inhibitory effects of LPS on GnRH secretion in ewes during the follicular phase of the estrous cycle. Intracerebroventricular administration of LPS at a dose of 400 ng/kg decreased GnRH gene expression in the preoptic area (POA) and the median eminence (ME). A reduction in GnRH peptide expression was also observed in the POA. These findings are consistent with our previous study, which showed that LPS suppresses hypothalamic GnRH secretion [30]. In our earlier work on anestrous ewes, endotoxin-dependent inflammation reduced GnRH mRNA expression, particularly in the POA, the hypothalamic structure where the majority of GnRH neurons are located [7]. The inhibitory effect of LPS-induced inflammation on GnRH synthesis is likely mediated by centrally acting proinflammatory cytokines, particularly IL-1β and TNFα [31,32]. These cytokines may be mainly of peripheral origin [33,34], although local synthesis within the hypothalamus is an additional important source [35]. Microglial cells are considered the primary site of proinflammatory cytokine synthesis within the central nervous system [36]. Although their role in disrupting hypothalamic secretory activity during endotoxin-induced inflammation seems to be unquestionable, changes in GnRH secretion may also be caused by the direct central action of LPS [7], as its molecules are partially able to cross the blood–brain barrier (BBB). Studies in rats [37] and mice [38] demonstrated that measurable amounts of iodine-radiolabeled LPS cross the BBB and reach the brain parenchyma. Evidence from rat studies also implies that LPS enters the brain under normal physiological circumstances, possibly through transport involving lipoproteins [38].

In the present experiment, we demonstrated that central administration of AEA prevented LPS-induced suppression of GnRH secretion in ewes during the follicular phase. This effect may result from a direct action of AEA on GnRH secretion or from its anti-inflammatory properties mediated through the modulation of cytokine activity. Our previous experiment on anestrous ewes showed that ICV injection of AEA at a concentration of 30 µM elevated GnRH mRNA expression in the ME, but not in the POA. However, AEA exerted a stimulatory effect on GnRH peptide content in both the POA and ME [26]. In contrast, studies conducted primarily in rats have shown that cannabinoids (CBs) and endocannabinoids (ECBs) generally exert an inhibitory effect on the HPG axis. Scorticati et al. [39] demonstrated that AEA suppressed GnRH release from the MBH in male rats, while it had no effect on hypothalamic GnRH secretion in ovariectomized (OVX) females. However, in vitro studies using MBH tissue from estrogen-supplemented OVX rats (OVX-E) demonstrated that AEA stimulated GnRH release, whereas no such effect was observed in OVX rats without estrogen administration. These findings suggest that estrogen may reverse the inhibitory effect of AEA on GnRH secretion in female rats. A potential mechanism for this inhibition involves gamma-aminobutyric acid (GABA), a key neurotransmitter regulating GnRH secretion. Fernandes-Solari et al. [40] demonstrated that AEA incubation significantly increased GABA release from the MBH of male rats in vitro. Since CB1Rs are expressed on GABAergic interneurons in the hippocampus and neocortex [23], and GABA generally inhibits GnRH secretion, increased GABA release may contribute to reduced GnRH output. In ewes, GABA(A) receptors also play a significant role in regulating GnRH secretion, although the underlying neural mechanisms may differ. The inhibitory effects of GABA may be mediated through interactions with β-endorphinergic and catecholaminergic systems, whose activity depends on the animal’s physiological state [41]. Cannabinoids have been shown to influence catecholaminergic and serotonergic systems, both of which are involved in the hypothalamic regulation of GnRH secretion. ICV administration of AEA has been shown to increase hypothalamic norepinephrine levels in mice [42]. Given the significant role of the dopaminergic system in regulating GnRH secretion in ewes [43], its interaction with cannabinoids is of particular relevance. Scorticati et al. [39] showed that cannabinoids can affect brain dopamine (DA) levels and dopamine receptor expression. Their study further demonstrated that AEA stimulates both the synthesis and release of DA from MBH explants, as evidenced by an increased ratio of 3,4-dihydroxyphenylacetic acid (DOPAC) and DA, indicating accelerated DA turnover. Interestingly, the effect of AEA on dopaminergic neuronal activity differed between male and female rats that were either OVX or OVX-E [39]. These findings imply that estrogen may stimulate AEA release from MBH neurons, which then acts on cannabinoid receptors located on tuberoinfundibular DA neurons to inhibit DA release. This hypothesis is supported by the observation that CB1R colocalizes with the cell bodies of periventricular tyrosine hydroxylase-containing neurons, DA neurons that project to the ME, where DA can regulate GnRH secretion [44]. Overall, the impact of AEA on GnRH release appears to stem from complex interplay between the endocannabinoid system and neurotransmitters such as catecholamines.

Unexpectedly, in the present study, ICV administration of AEA did not affect LPS-inhibited LH concentrations. The only effect was that LPS reduced LHβ gene expression in the AP, and this effect was not reversed by ICV administration of AEA. It is important to emphasize that LH was assessed at 2–3 h post—LPS treatment and that pulsatile secretion of this hormone defines feature of LH dynamics. To fully understand changes in LH secretion the serial blood sampling and pulse analysis is needed. Our previous research on follicular-phase ewes demonstrated that LPS suppressed LH secretion [3]. Similarly, inhibitory effects of bacterial endotoxin have been reported in anestrous ewes, where LPS diminished both LHβ gene expression and LH release [45]. Disturbances in LH secretion in the AP accompanying inflammation appear to result mainly from reduced GnRH secretion in the hypothalamus. However, the changes in the secretory activity of the pituitary may also result, at least partially, from the direct action of LPS on this gland [46,47]. Here, central (ICV) administration of AEA did not reverse the LPS-induced decrease in LHβ gene expression in the AP. This contrasts with numerous rat studies demonstrating that both exogenous (e.g., THC) and endogenous (e.g., AEA) cannabinoids inhibit gonadotropin release. For instance, chronic supply of cannabinoids in prepubertal female rats reduced serum LH and sex steroid levels [48], while acute THC treatment inhibited pulsatile LH secretion and blocked estrogen- or estrogen/progesterone-induced LH surge in ovariectomized rats [49]. In our previous in vitro study employing AP explants from anestrous ewes, AEA also directly suppressed LH secretion [50]. However, that model involved direct exposure of AP cells to AEA, while the present in vivo approach allowed AEA to act through integrated pathways at both hypothalamic and pituitary levels. These results may reflect incomplete restoration of HPG axis signaling at the pituitary level. On the one hand, the lack of effect of AEA on LH release during immunological stress may be due to the reduced level of GnRHR gene expression in the AP. We found that LPS-induced inflammation decreased GnRHR gene expression in the ME and AP. AEA had no impact on the LPS-induced reduction in GnRHR gene expression. The above-mentioned studies ex vivo study on ovine AP explants demonstrated that AEA could inhibit LH secretion by downregulating GnRHR [50]. On the other hand, in our study, AEA was administered directly into the hypothalamus region and its anti-inflammatory effect was found in this area. However, the administration of LPS induces a strong inflammatory response, including significantly increasing the level of proinflammatory cytokines circulating in the blood, as shown in animal studies and also in humans [51,52]. Circulating inflammatory mediators can directly affect processes occurring in the pituitary, because this gland sits outside the blood–brain barrier. The study on rodents showed that both posterior and anterior pituitary gland expressed large number of IL-1 receptors and suggested pituitary cells may be a direct target for IL-1 [53]. Our recent findings revealed that the ovine anterior pituitary expresses considerable levels of mRNA for proinflammatory cytokines, including IL-1β, IL-6, and TNFα, along with their respective receptors [10]. The expression of cytokine receptors by pituitary cells suggests that these ligands can modulate the gland’s hormonal secretory functions. In support of this, our previous ex vivo experiment demonstrated that IL-1β inhibited LH secretion and decreased GnRHR gene expression in GnRH-stimulated ovine pituitary explants [8]. Such direct pituitary effects of inflammatory mediators like IL-1β may underlie the lack of LH response despite the AEA-induced stimulation of hypothalamic GnRH secretion.

Another mechanism by which AEA may counteract the LPS-induced suppression of GnRH/LH secretion is through its anti-inflammatory properties, mediated by the modulation of cytokine activity. Exogenous, endogenous, and synthetic cannabinoids are widely recognized as immune regulators [27]. Here, AEA reduced LPS-stimulated IL-1β concentration at the hypothalamic level, although no changes in gene expression were observed. We also found that AEA increased IL-1Ra gene expression in hypothalamic structures. Interestingly, IL-10 concentrations were significantly elevated after AEA treatment compared with control and LPS-treated ewes. These findings suggest that the antiinflammatory action of central AEA may rely on the suppression of proinflammatory mediators such as IL-1β, consistent with previous studies showing similar effects following peripheral AEA administration during endotoxin-induced inflammation [54]. Additionally, AEA treatment has been shown to reduce serum levels of the proinflammatory cytokine IL-1β in rats subjected to water immersion and restraint stress [55]. Cannabinoids also display anti-inflammatory properties in the brain in neurodegenerative diseases associated with neuroinflammation. In murine neurodegeneration models, cannabidiol has been shown to decrease CNS levels of proinflammatory cytokines, including IL-1β [56]. Similarly, in virus-infected mice, R(+)WIN55,212, a CB1 receptor agonist, significantly reduced mRNA expression of IL-1β, IL-6, and TNFα [57]. The anti-inflammatory action of central AEA observed in our study likely involves a dual mechanism: the suppression of proinflammatory mediators and the stimulation of anti-inflammatory cytokines like IL-10. This aligns with in vitro studies demonstrating that AEA promotes IL-10 production in microglial cells, primarily through CB2 receptor activation and downstream anti-inflammatory signaling pathways [58]. Therefore, the significant increase in IL-10 concentration following AEA treatment in our experiment is consistent with this established mechanism. Correa et al. [58] have demonstrated that AEA increases LPS/IFN-γ-induced IL-10 production in microglia by targeting CB2Rs and downstream ERK1/2 and JNK MAPKs signaling pathways. The relationship between cannabinoids and cytokines is bidirectional, as they can influence cytokine secretion, while cytokines can modulate cannabinoid receptor activity. This dynamic interplay has the potential to alter cytokine profiles from a proinflammatory to an anti-inflammatory state [59]. Stimulation of CB1R by synthetic cannabinoids suppresses the synthesis of proinflammatory cytokines in glial cells, demonstrating anti-inflammatory effects in vitro [60,61]. It should be emphasized that research conducted on a mouse model with CB1R deletion specifically in microglia, subjected to an immune challenge, indicated that the role of CB1R in regulating neuroinflammation is more complex than previously thought. The study revealed that microglial CB1R differentially modulates sickness behavior in males and females. In males, knocking out CB1R in CX3CR1-positive cells reduced IL-1β and TNF-α mRNA synthesis, whereas in females, microglial CB1R deletion had no significant effect on cytokine production in the hippocampus [62]. In our in vivo experiment we did not use CB1R and CB2R antagonists, so we cannot infer the role of CB1R vs. CB2R. Further studies with agonists and antagonists of individual receptors are required. Conversely, endocannabinoids such as AEA can serve as a source of arachidonic acid (AA), which is further metabolized by eicosanoid-synthesizing enzymes to generate bioactive lipids possessing pro- or anti-inflammatory functions. Pharmacological blockade of endocannabinoid degradation may increase the levels of these immunomodulatory lipids, consequently affecting the activity of inflammatory cells [63]. Arachidonic acid (AA) is a polyunsaturated fatty acid found in the phospholipids of cell membranes throughout the body and is particularly abundant in the brain. Through enzymatic conversion of AA, mediators such as prostaglandins, thromboxanes, and leukotrienes are synthesized de novo. One of these products is prostaglandin E₂ (PGE₂). In both the pituitary and hypothalamus, AA and its metabolites play important roles in regulating the secretion of various peptide hormones [64]. A few studies have demonstrated that central administration of the phospholipase A₂ activator melittin, arachidonic acid (AA), or thromboxane A₂ can stimulate the catecholaminergic, vasopressinergic, and renin–angiotensin systems [65,66]. These neuropeptides are capable of modulating GnRH and LH secretion. Moreover, numerous studies have demonstrated that PGE₂ serves as an important regulator of GnRH release [67]. Additionally, intracerebroventricular administration of AA was shown to elevate plasma FSH, LH, and testosterone concentrations in conscious male rats [68]. Additionally, the anti-inflammatory effect of AEA in the CNS may involve increased expression of IL-1Ra and IL-1R2 genes [69].

It is worth mentioning that our study showed that LPS treatment significantly elevated the circulating level of cortisol, whereas this stimulatory effect had been reduced in AEA-treated animals. An increase in circulating glucocorticoids during stress is thought to inhibit the secretion of GnRH and gonadotropins. In sheep, cortisol has been shown to act at the pituitary level to decrease its responsiveness to GnRH and to lower LH pulse frequency. Moreover, cortisol suppresses GnRH pulse frequency in follicular-phase ewes, an effect that appears to depend on the presence of ovarian steroids [70]. Nonetheless, accumulating evidence suggests that the impact of stress on gonadotropin secretion is not straightforward. Recent findings indicate that acute stress may activate rather than inhibit the hypothalamic–pituitary system. In rats, acute stress has been shown to accelerate and intensify the preovulatory LH surge [71]. It has been proposed that activation of corticotropin-releasing hormone (CRH) receptors, particularly CRHR1, may enhance LH secretion under specific conditions. Endogenous cannabinoids are key regulators of both the physiological and behavioral aspects of stress responses. It is also considered that through buffering basal activity and mediating fast glucocorticoid feedback, endocannabinoid signaling contributes to the maintenance of HPA axis homeostasis [72]; therefore, decreased circulating cortisol concentration after AEA treatment is not surprising. We cannot exclude that activation of the HPA axis and interaction of AEA with glucocorticoid secretion influenced GnRH/LH secretion, but this requires further in-depth research.