Effect of Electro-acupuncture: α (1)- and β (2)-adrenoceptors and p75 Neurotrophin Receptors
Effect of Electro-acupuncture on Ovarian Expression of α (1)- and β (2)-adrenoceptors, and p75 Neurotrophin Receptors in Rats with Steroid-induced Polycystic Ovaries
An
Background
Estradiol valerate (EV)-induced polycystic ovaries (PCO) in rats is associated with an increase in ovarian sympathetic outflow. Low-frequency (2 Hz) electro-acupuncture (EA) has been shown to modulate sympathetic markers as well as ovarian blood flow as a reflex response via the ovarian sympathetic nerves, in rats with EV-induced PCO.
Methods
In the present study, we further tested the hypothesis that repeated 2 Hz EA treatments modulate ovarian sympathetic outflow in rats with PCO, induced by a single i.m. injection of EV, by investigating the mRNA expression, the amount and distribution of proteins of α1a-, α1b-, α1d-, and β2-adrenoceptors (ARs), as well as the low-affinity neurotrophin receptor (p75NTR).
Results
It was found that EV injection results in significantly higher mRNA expression of ovarian α1b- and α1d-AR in PCO rats compared to control rats. The p75NTR and β2-ARs mRNA expression were unchanged in the PCO ovary. Low-frequency EA resulted in a significantly lower expression of β2-ARs mRNA expression in PCO rats. The p75NTR mRNA was unaffected in both PCO and control rats. PCO ovaries displayed significantly higher amount of protein
Conclusion
The present study shows that EA normalizes most of the EV-induced changes in ovarian ARs. Furthermore, EA was able to prevent the EV-induced up regulation of p75NTR, probably by normalizing the sympathetic ovarian response to NGF action. Our data indicate a possible role of EA in the regulation of ovarian responsiveness to sympathetic inputs and depict a possible complementary therapeutic approach to overcoming sympathetic-related anovulation in women with PCOS.
Introduction
Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine and metabolic disorder
The precise etiology of the disease is unknown, even though the disturbances detected in PCOS has been attributed to primary defects in the hypothalamus-pituitary-adrenal (HPA) axis, the ovarian microenvironment, the adrenal gland, and the insulin/insulin-like growth factor (IGF)-I metabolic regulatory system [1]. That the sympathetic nervous system may be a primary factor in the development and maintenance of PCOS has been suggested by several investigators [2-5].
The utility of murine models of polycystic ovaries (PCO) has been discussed [6]. Even though it is impossible to reproduce human PCOS in an animal model, such a model may provide important leads. Studies on adult normal cycling rats found that a single intramuscular (i.m.) injection of estradiol valerate (EV) causes acyclicity and formation of PCO [7]. The EV-induced rat PCO model reflects some endocrinological and morphological characteristics of human PCOS, and it is assumed that activity in the ovarian sympathetic nerves is higher than in normal rats [8-10]. This is evidenced by an early increase in ovarian levels of norepinephrine (NE), an enhanced release of NE from ovarian nerve terminals, an increased activity of the catecholamine synthesis-limiting enzyme tyrosine hydroxylase (TH), and down-regulation of β2-adrenoceptors (ARs) in theca-interstitial cells [8-10].
The expression of other types of ARs in the ovary, namely the α1-ARs, has been evaluated by functional studies. α1-ARs are members of the G protein-coupled receptors and play critical roles in the regulation of a variety of physiological processes [11]. Within this classification, there are three subtypes: α1a, α1b, and α1d [11]. The α1a-AR subtype has been reported to be implicated in the maintenance of vascular basal tone, the α1b-AR subtypes to participate in the response to exogenous agonists, and that the α1d-AR subtype is a predominant mediator of arterial vasoconstriction. In vitro studies have demonstrated that α-AR are involved in the regulation of ovarian blood flow [12] and most probably in the ovarian steroidogenesis [13]. In a recent study, we found that the expression of all the α1-AR subtypes in the ovaries of PCO rats significantly differs from that of controls and varies at different time points after EV injection, indicating a possible participation of this ARs in the development of EV-induced PCO [14].
It has been demonstrated that the development of ovarian follicular cysts in steroid-induced PCO in rats is preceded by an increased synthesis of ovarian nerve growth factor (NGF) and low-affinity neurotrophin receptor (p75NTR) mRNA [10]. Thus, blocking the actions of intra-ovarian NGF restores estrus cyclicity as well as structural and functional features of the ovary in EV-induced PCO in rats [10], suggesting that hyper activation of sympathetic input in PCO is related to an overproduction of NGF.
Electro-acupuncture (EA) is a non-pharmacological method known to initiate a number of reactions at the spinal level and centrally in the brain [15,16]. We have recently demonstrated that repeated low-frequency EA treatments induced regular ovulations in more than one-third of the women affected by PCOS and normalized endocrine and neuroendocrine parameters without any negative side-effects [17]. These observation suggest that EA effects are mediated through inhibition of the activity of the sympathetic nervous system since EA is known to modulate various autonomic functions [17]. Moreover, using the steroid-induced PCO model, we found that repeated treatments of low-frequency EA in somatic segment related to the innervation of the ovary, reduced high concentrations of ovarian NGF, corticotrophin-releasing factor (CRF), and endothelin-1 as well as increased low concentrations of hypothalamic β-endorphin [18-21]. Furthermore, low-frequency EA increases ovarian blood flow as a reflex response via the ovarian sympathetic nerves, whereas high frequency decreases ovarian blood flow as a passive response following systemic circulatory changes in both normal and PCO rats [22,23]. These results suggest that repeated treatments of low-frequency, but not high frequency EA, can inhibit high activity in the autonomic nervous system. However, the mechanism implicated in this event is not clearly known.
The present study was undertaken to investigate the effect of repeated treatments of low-frequency EA on ovarian sympathetic innervation in rats with steroid-induced PCO. To address this question, we studied the mRNA expression and protein amount and distribution of the sympathetic markers α1a-, α1b-, α1d-, and β2-AR, and of p75NTR.
Materials and methods
Animals
Thirty-two virgin adult cycling Wistar Kyoto rats (Mllegaard, Denmark) weighing 205-230g were housed four to a cage at a controlled temperature of 22°C with a 12-h light:12-h dark cycle for at least 1 week before and throughout the experimental periods. The rats had free access to pelleted food and tap water. Sixteen rats, those in the two PCO groups described below, were each given a single i.m. injection of 4 mg EV (Riedeldehaen, Germany) in 0.2 ml oil, to induce well-defined PCO [7,18]. Sixteen rats, those in the two Oil groups described below, received a single i.m. injection of 0.2 ml oil (arachidis oleum, Apoteket AB, Ume, Sweden) only. Thirty to thirty-three days after i.m. injection of EV, i.e. 2 days after the last EA treatment, the rats was killed by decapitation. The injections and the finalizing of the experiment was done independent of cycle day [7,18]. The experiments were carried out according to the principles and procedures outlined in the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the local animal ethics committee at Gteborg University, Gteborg, Sweden
Electro-acupuncture treatment
The rats were divided into four experimental groups: i) an Oil group (control, n = 8), ii) an Oil group receiving EA (EA, n = 8), iii) a PCO group (PCO, n = 8), and iv) a PCO group receiving EA (PCO+EA, n = 8).
Internal server error