Neuropeptides (2001) 35(5&6), 227±231

ß 2001 Elsevier Science Ltd

doi: 10.1054/npep.2002.0878, available online at http://www.idealibrary.com on

Effects of electro-acupuncture on
corticotropin-releasing factor in rats with
experimentally-induced polycystic ovaries
E. Stener-Victorin,1 T. Lundeberg,2 U. WaldenstroÈm,1
I. Bileviciute-Ljungar,2 P. O. Janson1
1
Department of Obstetrics and Gynecology, GoÈteborg University, GoÈteborg, Sweden, 2Department of Physiology and
Pharmacology, Karolinska Institutet, Stockholm, Sweden

Summary The aims of the present study were to investigate corticotropin-releasing factor (CRF) concentrations in the
brain, the adrenal glands, and the ovaries in rats with estradiol valerate (EV) induced polycystic ovaries (PCO). The
effect of 12 electro-acupuncture (EA) treatments on CRF concentrations was also investigated. The CRF
concentrations in the median eminence (ME) were significantly increased in rats with PCO (both the PCO control
group and the PCO group receiving EA) compared with the healthy control group (veichle control group), indicating
increased activity in the hypothalamus-pituitary axis. The CRF concentrations in the ovaries were significantly reduced
in the PCO group receiving EA compared with the PCO control group. Also, there was a decrease in comparison
with the healthy control group but the decrease was not as significant. This finding indicates that repeated EA
treatments change the neuroendocrinological state in the ovaries, which may play an important role in reproductive
failure. ß 2001 Elsevier Science Ltd. All rights reserved

INTRODUCTION The sympathetic nervous system is known to regulate

ovarian function and could act as a potential source of
Alterations in the neuroendocrine system caused by stress
CRF delivery within the ovary (Ojeda and Lara, 1989).
( physical or emotional) can induce pathological changes
CRF has also been identi®ed in the rat spinal cord and
in reproductive functions, such as anovulation (Rivest and
may be released locally by ®bres innervating blood
Rivier, 1993; Rivier and Rivest, 1991). Corticotropin-
vessels, interstitial tissues, and developing follicles. In the
releasing factor (CRF) is a stress-related peptide, which is
ovaries CRF may regulate ± as do adrenergic agents ±
produced in the hypothalamic paraventricular nucleus
follicular development, steroid secretion, and ovulation.
and is released into hypothalamo-pituitary-adrenal (HPA)
Although little is known, it is tempting to suggest that
axis. CRF has also been identi®ed in several peripheral
there are functional interactions between the activity of
tissues including the ovary (Mastorakos et al., 1994;
the sympathetic nervous system and the ovarian CRF-
Mastorakos et al., 1993; Rivest and Rivier, 1993). The anti-
ergic system (Nappi and Rivest, 1995). Recent ®ndings
reproductive effect of this peptide as well as endogenous
support this suggestion by the presence of CRF in the
opioids released under stress has been extensively studied
peripheral nervous system of the rat (Bileviciute et al.,
at various levels of the HPA and the hypothalamic-
1997; Veraksits et al., 2000).
pituitary-gonadal (HPG) axis (Nappi and Rivest, 1995).
Sensory stimulation, i.e. electro-acupuncture (EA),
stimulates/modulates the release of b-END and other pep-
Received 30 December 2000
tides into the blood and the nervous system (Andersson
Revised 23 July 2001
Accepted 20 September 2001 and Lundeberg, 1995; UvnaÈs-Moberg et al., 1993). These
hormones reach different target organs and play import-
Correspondence to: Elisabet Stener-Victorin, Department of Obstetrics and
Gynecology, Kvinnokliniken, Sahlgrenska University Hospital, SE-413 45
ant roles in stress responses and may also affect central
GoÈteborg, Sweden. Tel.: ‡ 46 31 342 3338; Fax: ‡ 46 3182 9248; autonomic out¯ow by regulating the vasomotor centre in
E-mail: elisabet.stener-victorin@medstud.gu.se the brainstem. EA may also regulate sympathetic out¯ow

227
228 Stener-Victorin et al.
at a spinal level by stimulating muscle afferents in somatic
segments corresponding to the affected organ (Andersson,
1993; Andersson and Lundeberg, 1995; Sato et al., 1997).
In addition, it is possible that EA exerts a peripheral effect
by modulating the release of neuropeptides from periph-
eral nerve endings ( Jansen et al., 1989; Kaada et al., 1984;
Lundeberg et al., 1991; Sato et al., 2000). An experimentally-
induced polycystic ovary (PCO) rat model ± produced by a
single intramuscular (i.m.) injection of estradiol valerate
(EV) (Brawer et al., 1986) was used to study the effects of
EA by analysing CRF in the central nervous system and
endocrine organs.
Thus, the aim of the present investigation was to study
CRF tissue concentrations in the brain, adrenals, and
ovaries and the effects of EA on rats with experimentally-
induced PCO associated with anovulation and a hyper-
active sympathetic nervous system.
MATERIALS AND METHODS
Thirty-four virgin adult cycling Sprague±Dawley rats
(MoÈllegaard, Denmark) weighing 190±210 g, with regular
4-day oestrus cycles were used. They were housed four to
a cage with free access to pelleted food and tap water and
at a controlled temperature of 228C with a 12-h light/12-h
dark cycle for at least 1 week before and throughout the ex-
perimental period. The 17 rats in the PCO groups were
each given a single i.m. injection of 4 mg EV (Riedeldehaen,
Germany) in 0.2 ml oil to induce well-de®ned PCO (Brawer
et al., 1986). The 17 rats in the group of healthy controls
were each given 0.2 ml oil alone. All rats were anaesthe-
tized super®cially with en¯uran (EFRANEt, Abbott
Scandinavia, Kista, Sweden) before decapitation on day
30 after i.m. injection, i.e. 1±2 days after the last treatment.
The study was approved by the local Ethics Committee for
Animals at GoÈteborg University.
EA treatment
The rats were divided into three experimental groups: (i)
healthy control group (vehicle control) (n ˆ 17); (ii) PCO
control group (n ˆ 8); and (iii) PCO group receiving EA
(n ˆ 9). All groups were anaesthetized during 25 min, 12
times, corresponding to the EA treatment given to the PCO
group receiving EA. The anaesthesia was induced by in-
halation of en¯uran at a rate of 5.5±6.5 ml/h, with an O2
and air ¯ow of 0.25 l/min. The PCO group receiving EA
was given EA during anaesthesia every second or third day
beginning 2 days after EV injection. The points chosen for
stimulation were in somatic segments corresponding to
the innervation of the ovaries (Th 12-L2, S2-S4) and bilat-
eral in mm biceps femoris and erector spinae. The needles
(Hegu: Hegu AB, Landsbro, Sweden) were inserted to
depths of 0.5±0.8 cm and then attached bilaterally to an
Neuropeptides (2001) 35(5&6), 227±231
electrical stimulator (CEFAR ACU II, Cefar, Lund, Sweden)
and stimulated with a low-burst frequency of 2 Hz.
Individual pulses within the burst frequency were square
wave pulses with alternating polarities and pulse dur-
ations of 0.2 ms, 80 pulses per second. The intensity was
adjusted until local muscle contractions were seen to re-
¯ect the activation of muscle-nerve afferents (A-delta
®bres and possibly C ®bres) (Haker and Lundeberg,
1990; Lundeberg et al., 1988). The location and type of
stimulation were the same in all rats.
CRF measurements by radioimmunoassay (RIA)
After the rats were decapitated, the median eminence
(ME), the hypothalamus, the hippocampus, one ovary,
and one adrenal were quickly removed and dissected on
dry ice, weighed, and stored at 808C until extraction.
CRF-like immunoreactivity (CRF-LI) was analysed by
using commercial antibodies for human/rat CRF
(Phoenix Pharmaceuticals, Inc. California, USA). High
Performance Liquid Chromatography (HPLC)-puri®ed
I125-Histidyl human/rat CRF was used as a radioligand,
and human/rat CRF (Neosystem, France) as a standard.
The lower detection limit for CRF-LI was 7.8 pmol/L.
Statistical analyses
Statistical analyses were made using SPSS 8.0 software.
CRF concentrations in the ME, the hypothalamus, the
hippocampus, the ovary, and the adrenal glands were
analysed using ANOVA, and the groups were tested
using multiple comparisons with the correction of
Bonferroni. Due to the lack of signi®cance in Levene's
test of homogeneity of variances for the ME and the
ovary, the ordinary t-test was used instead. All results are
given as mean + standard error of mean (SEM). A P-value
less than 0.05 was considered signi®cant. The 95% con®-
dence interval (CI) was given when P < 0.05.
RESULTS
CRF
Means + SEM for CRF concentrations (pmol/g wet weight)
in the ME, the hypothalamus, the hippocampus, the ovary,
and the adrenal glands in all groups are presented in
Figures 1 and 2.
The CRF concentrations in the ME were signi®cantly
higher in both the PCO control group (P < 0.01,
CI ˆ 56.1, 209.2) and the PCO group receiving EA
(P < 0.05, CI ˆ 1.4, 170.0) than in the healthy control
group ( Fig. 1). The CRF concentrations in the ovaries
were signi®cantly lower in the PCO group receiving
EA than in both the PCO control group (P < 0.01,
ß 2001 Elsevier Science Ltd. All rights reserved.
 Effects of electo-acupuncture on corticotropin-releasing factor in rats 229

pmol/g ME Ovary
pmol/g *a
250 ** a 0.14
** b
200 *b 0.12
0.10
150
0.08
100 0.06

50 0.04
0.02
0
Healthy control PCO control PCO + EA 0.00
Healthy control PCO control PCO + EA
pmol/g Hypothalamus
pmol/g Adrenal gland
12
0.30
10
0.25
8
0.20
6
0.15
4
0.10
2
0.05
0
0.00
Healthy control PCO control PCO + EA
Healthy control PCO control PCO + EA

pmol/g Hippocampus Fig. 2 CRF (pmol/g) in the ovary and the adrenal gland. All data
values are mean + SEM. Significance *P < 0.05, **P < 0.01;
0.60 a ˆ healthy control versus PCO ‡ EA, b ˆ PCO control versus
0.50

0.40

0.30 concentrations in the ME compared to healthy controls

0.20 and that repeated EA treatments result in signi®cantly
lower ovarian CRF concentrations in PCO rats.
0.10 CRF is the principal neurohormone in the control of the
0.00 HPA and HPG axes. The ®ndings in the present study of
Healthy control PCO control PCO + EA increased CRF concentrations in the ME in both PCO
Fig. 1 CRF (pmol/g) in the median eminens, the hypothalamus, and groups compared to in the healthy control group indicates
the hippocampus. All data values are mean + SEM. Significance an involvement of these axes in the experimentally-
*P < 0.05, ** P < 0.01; a ˆ healthy control versus PCO control,
induced PCO model (Gindoff and Ferin, 1987). It is un-
b ˆ healthy control versus PCO ‡ EA.
likely that these changes are caused by the anaesthesia
since the healthy control group were anaesthetized as
well. However, it does indicate that a single injection of
estradiol valerate induces a stressed state in PCO rats with
CI ˆ 0.03, 0.13) and the healthy control group (P < 0.05, a possible in¯uence on the HPA and HPG axes. The alter-
CI ˆ 0.11, 0.008) (Fig. 2). ations in ME concentrations in both PCO groups were not
The CRF concentrations in the hypothalamus, the affected by repeated EA treatments.
hippocampus, and the adrenals were not signi®cantly dif- The presence of CRF and its receptors in normal rat
ferent between the groups. ovaries has previously been demonstrated and raises the
question of local involvement of CRF in ovarian physi-
ology (Gindoff and Ferin, 1987; Mastorakos et al., 1994;
DISCUSSION
Mastorakos et al., 1993). First, ovarian CRF might act as a
The main ®ndings in the present study were that experi- bioactive cytokine during ovulation and luteolysis and
mentally-induced PCO is associated with increased CRF might also be involved in follicular atresia (Murdoch

ß 2001 Elsevier Science Ltd. All rights reserved. Neuropeptides (2001) 35(5&6), 227±231
230 Stener-Victorin et al.
et al., 1988). Second, CRF might participate in ovarian
steroid biosynthesis, in analogy to its effect on testosterone
biosynthesis by Leydig cells (Fabbri et al., 1990; Ulisse
et al., 1989). Interestingly, the primary oocytes of primor-
dial follicles in the ovaries of women with PCOS ± but not
in the ovaries of women without PCOS ± have been found
to contain CRF (Mastorakos et al., 1994). This ®nding sug-
gests that the local involvement of CRF in the ovary may
be relevant to oocyte dysfunction and reproductive failure
in women with PCOS (Chrousos et al., 1993; Mastorakos
et al., 1994).
The most interesting ®nding in the present study is that
repeated EA treatments signi®cantly decreased ovarian
CRF concentrations in the PCO group compared with
both the PCO control and the healthy control groups.
There is much evidence that the present rat PCO model
is associated with hyperactivity in the ovarian sympa-
thetic nerves, shown by an overproduction of nerve
growth factor (NGF) (Barria et al., 1993; Dissen et al.,
2000; Lara et al., 2000; Lara et al., 1993). It has also been
suggested that activation of this neurotrophic-neurogenic
regulatory loop is a component of the pathological process
by which EV induces cyst formation and anovulation in
rats (Dissen et al., 2000; Lara et al., 2000). There was no
difference in CRF concentration between the untreated
PCO control group and the healthy control group in the
present study. The lack of changes in the PCO control
group makes it unlikely that an alteration in peripheral
CRF delivered to the ovary via the nerves plays a crucial
role in the pathological process of experimentally-induced
PCO.
However, the decrease in CRF concentrations in the
ovaries and the absence of an effect on CRF concentra-
tions in brain tissue after repeated EA treatments in the rat
PCO model used indicate that the EA effect may be attrib-
uted to a peripheral action on CRF. Recently, we demon-
strated that repeated EA treatments signi®cantly reduce
high ovarian NGF concentrations in rats with experimen-
tally induced PCO, which support the hypothesis that
repeated EA treatments inhibit an increased sympathetic
tone (Stener-Victorin et al., 2000a).
We and others have shown that repeated EA treatments
exert a `normalizing' effect on endocrinological and neu-
roendocrinological disturbances, as well as on anovula-
tion in women with PCOS (Chen, 1997; Chen and Yu,
1991; Gerhard and Postneek, 1992; Stener-Victorin et al.,
1996; Stener-Victorin et al., 2000b). The effect of repeated
EA has then been attributed to an inhibition of an over-
active autonomic nervous system.
Altogether, the result of the present study indicates an
in¯uence on the HPA and HPG axes in rats with experi-
mentally induced PCO. The decreased ovarian CRF con-
centrations following repeated treatments indicate a
peripheral EA action.
Neuropeptides (2001) 35(5&6), 227±231
ACKNOWLEDGEMENTS
The authors would like to thank Professor Owe Lundgren
and laboratory assistant Britt-Marie Fin, Department of
Physiology, GoÈteborg University, for providing excellent
working facilities and for invaluable laboratory help at the
Department. We wish to thank laboratory assistants Maud
Hoffstedt and Anja Finn, Department of Physiology and
Pharmacology, Karolinska Institutet, Stockholm, for their
invaluable help with the CRF analyses.
This study was supported by grants from the Hjalmar
Svensson foundation, Wilhelm och Martina Lundgrens
Vetenskapsfond (Wilhelm and Martina Lundgren's
Science Fund) and the Foundation for Acupuncture and
Alternative Biological Treatment Methods.
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