Effect of electro-acupuncture on ovarian expression of alpha (1)- and beta (2)-adrenoceptors, and p75 neurotrophin receptors in rats with steroid-induced polycystic ovaries

Abstract

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 alpha1a-, alpha1b-, alpha1d-, and beta2-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 alpha1b- and alpha1d-AR in PCO rats compared to control rats. The p75NTR and beta2-ARs mRNA expression were unchanged in the PCO ovary. Low-frequency EA resulted in a significantly lower expression of beta2-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 of alpha1a-, alpha1b- and alpha1d-ARs, and of p75NTR, compared to control rats, that were all counteracted by repeated low-frequency EA treatments, except for alpha1b-AR.

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.

Figure 1

Schematic drawing of the placement and stimulation of the acupuncture needles. Two needles were placed bilaterally in m. erector spinae at the level of Th12 and two were placed in m. quadriceps bilaterally. The needles were then attached to an electrical stimulator for electro-acupuncture (EA) treatment. Reprinted with permission from Biol Reprod (2000) 63:1507-1513.

Figure 2

Ovarian α_1a-AR mRNA and protein expression. As shown in panel A, no significant differences were found in α_1a-AR mRNA expression between the control, EA, and PCO groups. Values are given as means ± SEMs normalized to GAPDH. The results of α_1a-AR immunopositive cell count is shown in panel B. Values are given as means ± SEMs. Immunostaining revealed that ovarian α_1a-AR protein is expressed in control group ovaries. EA treatments did not affect the number of immunopositive cells in the ovaries of control rats. PCO ovaries had significantly higher amounts of α_1a-AR protein compared with control ovaries. EA treatments in PCO rats decreased α_1a-AR protein immunoreactivity when compared with untreated PCO rats. ^ap < 0.05 vs control group. ^bp < 0.05 vs PCO group. Representative pictures showing the distribution of α_1a-AR positive cells in the ovaries of the experimental groups are showed in Panel: C-D. Immunostained cells (arrows) were localized around blood vessels (C) and in the granulosa cells of an early antral follicle (D) and corpora lutea. F: Follicle; bv: blood vessel; gr: granulose cells; th: thecal layer. Magnification C: ×400; D: ×200.

Figure 3

Ovarian α_1b-AR mRNA and protein expression. As shown in panel A, no significant differences were found in α_1b-AR mRNA between the control and EA groups. A significant increase in ovarian α_1b-AR mRNA was found in the PCO group when compared to controls. No differences were found between the PCO+EA group and the PCO group. Values are given as means ± SEMs normalized to GAPDH. ^ap < 0.05 vs control group. EA treatments did not affect the amount of α_1b-AR immunopositive cells in the EA group(panel B). Significantly higher number of immunopositive cells of α_1b-AR was found in the ovaries of PCO rats. EA treatments did not affect the amount or distribution of α_1b-AR protein in PCO ovaries. Values are given as means ± SEM. ^ap < 0.05 vs control group. Representative pictures showing the distribution of α_1b-AR positive cells in the ovaries of the experimental groups are showed in Panels C-D. Immunostained cells (arrows) were localized around blood vessels (C) and in the granulosa cells of mature follicles (D). F: Follicle; bv: blood vessel; gr: granulose cells; th: thecal layer. Magnification C: ×400; D: ×200.

Figure 4

Ovarian α_1d-AR mRNA and protein expression. As shown in panel A, α_1d-AR mRNA expression was significantly higher in the PCO group than in control and EA group. EA significantly decreased the mRNA expression in the PCO+EA group compared with the PCO group. Values are given as means ± SEMs normalized to GAPDH. ^ap < 0.05 vs control group. The α_1d-AR immunopositive cell number (Panel B) was not affect in the EA group when compared to control group. Significantly higher number of immunopositive cells of α_1d-AR was found in the PCO group compared with the control group. EA treatment significantly decreased the number of immunopositive cells in the PCO+EA group. Values are given as means ± SEMs. ^ap < 0.05 vs control group. ^bp < 0.05 vs PCO group. Representative pictures showing ovarian distribution of α_1d-AR expressing cells (pointed by arrows) are showed in Panels C-D. The α_1d-AR was found expressed in the granulosa cells of healthy follicles (C) and corpora lutea (D) and around blood vessels (not shown) in all of the experimental groups. F: Follicle; gr: granulose cells; th: thecal layer. Magnification C-D: ×200.

Figure 5

Ovarian β₂-AR mRNA and protein expression. The expression of β₂-AR mRNA in the ovary (panel A) in the PCO group was lower compared to the control group. β₂-AR mRNA was unaltered in the PCO+EA group when compared to control. Values are given as means ± SEMs normalized to GAPDH. ^ap < 0.05 vs control group. β₂-AR immunopositive cell number (Panel B) in control ovaries was unchanged by EA treatments. No difference in number of β₂-AR immunoreactive cells was found in PCO ovaries, while EA treatments in PCO rats significantly increase the amount of β₂-AR immunostained cells when compared to PCO group. Values are given as means ± SEMs. ^bp < 0.05 vs PCO group. Representative pictures of stained cells (some of them pointed by black arrows) are showed in Panels C- D. The β₂-AR was found expressed in degenerating corpora lutea (C) and follicles (D) in all of the experimental groups. Magnification C: ×400; D: ×200.

Figure 6

Ovarian p75^NTR mRNA and protein expression. As shown in panel A, ovarian p75^NTR mRNA was found unchanged in the PCO group compared to the control and EA group. Low-frequency EA treatments did not significantly affect ovarian p75^NTRmRNA expression in the EA, PCO and PCO+EA groups. Values are given as means ± SEMs normalized to GAPDH. As shown in panel B, the number of ovarian p75^NTR immunopositive cells was significantly decreased in the EA group when compared to controls. The number of p75^NTR-stained cells in the PCO group was significantly higher than in controls, while repeated EA treatments greatly decreased p75^NTR protein immunoreactivity in the PCO+EA group. Values are given as means ± SEMs. ^ap < 0.05 vs control group. ^bp < 0.05 vs PCO group. Representative pictures of stained cells (pointed by black arrows) are showed in Panels C-D. The p75NTR was found expressed in the thecal layer of healthy follicles (C) and in the stromal region (D) in all of the experimental groups. Magnification C-D: ×200.