Polysaccharides from Wolfberry Prevents Corticosterone-
Induced Inhibition of Sexual Behavior and Increases
Benson Wui-Man Lau1,2,3, Jada Chia-Di Lee1,2,3, Yue Li3,4, Sophia Man-Yuk Fung1, Yan-Hua Sang1,
Jiangang Shen3,4, Raymond Chuen-Chung Chang1,2,3,5, Kwok-Fai So1,2,3,6*
1Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, People’s Republic of China, 2The State Key
Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, People’s Republic of China, 3Research Centre of Heart, Brain,
Hormone and Healthy Aging, Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, People’s Republic of China, 4School of Chinese Medicine, The
University of Hong Kong, Hong Kong SAR, People’s Republic of China, 5Laboratory of Neurodegenerative Diseases, Department of Anatomy, LKS Faculty of Medicine, The
University of Hong Kong, Pokfulam, Hong Kong SAR, People’s Republic of China, 6Joint Laboratory for Brain Function and Health (BFAH), Jinan University and The
University of Hong Kong, Guangzhou, People’s Republic of China
Lycium barbarum, commonly known as wolfberry, has been used as a traditional Chinese medicine for the treatment of
infertility and sexual dysfunction. However, there is still a scarcity of experimental evidence to support the pro-sexual effect
of wolfberry. The aim of this study is to determine the effect of Lycium barbarum polysaccharides (LBP) on male sexual
behavior of rats. Here we report that oral feeding of LBP for 21 days significantly improved the male copulatory
performance including increase of copulatory efficiency, increase of ejaculation frequency and shortening of ejaculation
latency. Furthermore, sexual inhibition caused by chronic corticosterone was prevented by LBP. Simultaneously,
corticosterone suppressed neurogenesis in subventricular zone and hippocampus in adult rats, which could be reversed
by LBP. The neurogenic effect of LBP was also shown in vitro. Significant correlation was found between neurogenesis and
sexual performance, suggesting that the newborn neurons are associated with reproductive successfulness. Blocking
neurogenesis in male rats abolished the pro-sexual effect of LBP. Taken together, these results demonstrate the pro-sexual
effect of LBP on normal and sexually-inhibited rats, and LBP may modulate sexual behavior by regulating neurogenesis.
Citation: Lau BW-M, Lee JC-D, Li Y, Fung SM-Y, Sang Y-H, et al. (2012) Polysaccharides from Wolfberry Prevents Corticosterone-Induced Inhibition of Sexual
Behavior and Increases Neurogenesis. PLoS ONE 7(4): e33374. doi:10.1371/journal.pone.0033374
Editor: Cesario V Borlongan, University of South Florida, United States of America.
Received July 20, 2011; Accepted February 13, 2012; Published April 16, 2012
Copyright: ? 2012 Lau et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the Azalea (1972) Endowment Fund, the University of Hong Kong Alzheimer’s Disease Research Network under Strategic
Research Theme on Healthy Aging and the Fundamental Research Funds for the Central Universities, no. 09ykpy25, 09ykpy31. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Lycium barbarum (commonly known as wolfberry) has been used as
an oriental herb in Asian countries for a long history . Being an
anti-aging herb,wolfberryhas been used formaintainingeyehealth,
nourishing the liver and kidney . Increasing lines of evidence
from both clinical and pre-clinical studies support the therapeutic
and health-promoting effects of wolfberry. A randomized, placebo-
controlled clinical study reveals the consumption of a juice prepared
from wolfberry promoted health like improving quality of sleep,
mental acuity and decreasing level of fatigue and stress . On the
other hand, different pre-clinical studies aim at determining the
precise biological activities of wolfberry and the active components
exerting the effect. Lycium barbarum polysaccharides (LBP), as a
major constituent of wolfberry , have been shown to exert a wide
range of biological effects, including neuroprotection against
neurotoxic insults [5,6], having anti-aging properties in an aging
animal model , prevention of glaucoma induced by elevated
intraocular pressure [4,8] and immune modulation .
Apart from the aforementioned effects, wolfberry was described
to exhibit pro-sexual effect by the Chinese herbalist Li Shizhen, and
thus wolfberry was included in sexual-enhancing Chinese herbal
remedies . Daily consumption of wolfberry juice improves the
well-being feeling towards sexuality, including increase in sexual
activity and ability . Several studies which focused on the
reproductive system showed that LBP was beneficial to male
reproduction in several aspects: first, the sexual performance of
hemicastrated rats could be improved by LBP treatment ;
second, the quality, quantity and motility of sperms were increased
 after LBP treatment;,third, damage of testis and seminiferous
epithelium by different insults, such as hyperthermia, H2O2and
testosterone of hemicastrated or irradiated rats were reversed by
LBP [11,12]. These findings provided evidence to support the
notion that LBP exerts therapeutic effects on male sexual
performance and fertility, although the mechanisms underlying
the effects remain largely unclear.
Successful sexual behavior involves complex interplay among
different systems including the reproductive system and the
nervous system . While previous studies about the effect of
LBP on sexual behavior focused on the reproductive system,
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there is scarce investigation of its influence on the nervous
system. The olfactory system and hippocampus in the brain are
important regions for perceiving sociosexual stimulus, processing
social memory and induce social recognition [13,14]. These two
regions are characterized by their continuous production of
adult-born neurons (neurogenesis) during the life. Increasing
lines of evidence suggests that neurogenesis may be involved in
sexual behavior [15,16]. For instance, increase in neurogenesis
in the subventricular zone (SVZ, a region produces new
neurons which migrate to the olfactory bulb) or hippocampus
could be found when a female experimental animal was
exposed to a male counterpart or sociosexual stimuli, which
could be observed in different species like prairie voles ,
mice  and ewes . If neurogenesis in the hippocampus
and SVZ are blocked, the normal sexual behavior will be
disrupted, including the mate-preference in female mice 
and male mating behavior of rats . Furthermore, the
newborn neurons were found to incorporate into the mating-
related brain circuit in hamsters . These studies suggest that
neurogenesis may have a reciprocal relationship with sexual
behavior and the newborn neurons may have an important role
in the regulation of sexual behavior.
Given the traditional reputation of wolfberry in promoting
sexual performance and the importance of neurogenesis in sexual
behavior, we ask 1. whether treatment of LBP could improve male
sexual behavior; 2. whether LBP could reverse sexual inhibition; 3.
whether LBP affects neurogenesis in in vivo and in vitro situations
and 4. if there is an association between LBP treatment, sexual
behavior and neurogenesis. The results will not only provide
evidence to support the pro-sexual functions of LBP, but also will
provide insight on the biological mechanisms underlying LBP
Materials and Methods
Animals and Treatment
The experimental protocol was approved by the animal ethics
committee of the University of Hong Kong (Committee on the
Use of Live Animals in Teaching and Research; protocol #1961–
09). Young adult male Sprague-Dawley rats (280610 g) were used
for the experiment. Animals were kept under a 12/12 light dark
cycle at 22uC. Sexually naı ¨ve animals were used since disruption of
sexual behavior is more obvious in sexually inexperienced animals.
Animals were randomly assigned to three experiments: (1) Effect of
LBP on sexual behavior at different dosages and time-points; (2)
subchronic treatment with corticosterone and LBP and (3) effect of
blocking neurogenesis in the pro-sexual effect of LBP. LBP was
prepared according to the procedures reported previously [4,8,20].
control group received daily oral feeding of vehicle solution
(0.01 M PBS). The other two groups of animals received either
1 mg/kg LBP or 10 mg/kg LBP feeding. The treatment continued
for 21 days with the sexual behavior test conducted at day 7, 14
and 21 of the treatment period (Figure 1A).
Animals were divided into three groups. The
control group received oral feeding of 0.01 M PBS and
subcutaneous sesame oil (vehicle of corticosterone treatment)
injection for 21 days. The corticosterone treatment group received
subcutaneous injection of 40 mg/kg corticosterone (Sigma, St
Louis, MO, USA; suspended in 0.8 mL sesame oil) and oral
feeding of PBS daily, while the co-treatment group received both
oral feeding of 1 mg/kg LBP and injection of corticosterone
during the treatment period. Bromodeoxyuridine (BrdU, Sigma,
Animals were divided into three groups. The
Figure 1. Treatment schedule of experiment 1 (A) and experiment 2 (B). (A): Animals were treated with 0, 1 or 10 mg/kg of LBP daily and
sexual behavior tests were conducted at 7-day intervals. (B): The three groups of animals received vehicle (PBS), corticosterone (40 mg/kg) or both
corticosterone and LBP treatment for 21 days. Sexual behavior test was conducted at day 21 and was followed by sacrifice of animals at day 22. (C):
After intracerebroventricular infusion of either saline (with PBS treatment) or Ara-c (with LBP treatment) for seven days, the animals were subjected to
sexual behavior test.
Wolfberry Improves Sexual Behavior
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St Louis, MO, USA) was injected intraperitoneally (50 mg/kg)
during the last three days of treatment at 12 h intervals (Figure 1B).
Animals were divided into two groups. The
control group received oral feeding of 0.01 M PBS and
intracerebroventricular infusion of normal saline. The Ara-c
group received oral feeding of 1 mg/kg LBP and infusion of
cytosine arabinoside (Ara-c, a cytostatic drug which blocks cell
proliferation) for seven days. Procedures of infusion was described
previously . In brief, osmotic pumps (Alzet, California, USA)
filled with saline/Ara-c were implanted subcutaneously at the
dorsal neck region of the rats. Saline/Ara-c was delivered via a
cannula, affixed at 1.5 mm lateral and 1.0 mm posterior, to the
bregma into the right lateral ventricle. At day 7, BrdU was injected
to label proliferative cells and the animals were subjected to sexual
behavior testing on the same day (Figure 1C).
Sexual Behavior Testing
Female rats (300620 g, stimulus animals) were induced to
become sexually receptive by subcutaneous injection of 50 mg of
estradiol benzoate 48 h before testing which was followed by
injection of 500 mg of progesterone 4 h before testing. The sexual
behavior test was started 2 hours after the onset of dark phase and
a dim red light source was used to illuminate the venue at 25–
40 lux. Before testing, the subject animals were placed in standard
rectangular plastic cages (25 (W) 6 28 (D) 6 24 (H) cm) for 10
minutes. Then, the sexually receptive females were introduced into
the cage and the mating process was video-recorded for 45
minutes for later analysis. The analysis of the sexual performance
was done by an experimenter blinded to the treatment conditions.
The components observed were: i. mount latency (ML, in
seconds): the time between introduction of the female rat and
the first mount; ii. intromission latency (IL, in seconds): the time
between introduction of the female rat and the first intromission;
iii. Mount frequency (MF): number of mount displayed before
ejaculation; iv. Intromission frequency (IF): number of intromis-
sion displayed before ejaculation; v. Copulatory efficiency (CE):
Total number of intromission divided by the sum of intromission
and mount frequency; vi. Ejaculation frequency (EF): number of
ejaculation during the test session and vii. Ejaculation latency (EL):
the time between the first intromission and ejaculation.
Tissue Processing and Immunohistochemistry
Animals were deeply anesthetized with an overdose of sodium
pentobarbital (100 mg/kg, i.p. injection), and transcardially
perfused with normal saline containing 4% paraformaldehyde.
After perfusion, the brains were dissected and post-fixed in 4%
paraformaldehyde overnight. Cryosections with thickness of
40 mm were prepared in 1-in-12 series using a freezing microtome.
Immunohistochemistry was performed as previous described
[22,23]. After affix on gelatin-coated slides, brain sections were
subjected to antigen retrieval (in 0.01 M citrate buffer, pH 6.0 at
85uC) for 25 minutes, followed by incubation in 2 N hydrochloric
acid at 37uC for 25 minutes. Then, the sections were incubated
with 0.1 M borax buffer (pH 8.5) for 15 minutes and subsequently
10% goat serum for block non-specific antigen binding. Primary
antibodies used were mouse anti-BrdU (1:1000, Roche, Indianap-
olis, IN, USA) and rabbit anti-doublecortin (DCX, an immature
neuronal marker, 1:300, Cell signaling technology, Beverly, MA,
USA). After incubation with primary antibody at room-temper-
ature overnight, sections were then incubated in diluted secondary
antibodies (goat anti-mouse Alexa Fluor 568 and goat anti-rabbit
Alexa Fluor 488, dilution 1:200, Molecular Probe, Eugene, OR,
USA) at room temperature for 2 h. Sections were examined under
a fluorescent microscope (Olympus Bx-52, Tokyo, Japan).
Quantification of BrdU-positive cells in the hippocampus
(sections from 2200 mm to 4800 mm posterior to bregma) and
subventricular zone (sections from 1800 mm anterior to bregma to
300 mm posterior to bregma) was performed using unbiased
stereology. Semi-automated StereoInvestigator (MicroBrightField,
Williston, VT, USA) was utilized. For the SVZ, results were
expressed as mean of BrdU-positive cells per section while
Figure 2. Treatment of LBP at 1 mg/kg improved male sexual
behavior in (A) Copulatory Efficiency, (B) Ejaculation Frequen-
cy and (C) Ejaculation Latency. No difference was found between
vehicle (PBS) group and LBP-treated group (10 mg/kg). Data are
presented as mean+SEM. *: p,0.05, ANOVA with LSD post-hoc test.
Wolfberry Improves Sexual Behavior
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estimated total number of BrdU-positive cells in the whole
structure was used for the hippocampus.
Enzyme-link Immunosorbant Assay of Testosterone in
Before transcardial perfusion, truncal blood of rats was collected
and heparinized. Plasma was isolated by centrifugation at 3000 g
for 15 minutes. The samples were stored at 280uC until the assay
was performed. Quantification of plasma testosterone level was
carried out using Testosterone EIA kit (Cayman, MI, USA)
according to the manufacturer’s protocol.
In Vitro Neurogenesis Assay
C17.2 neural stem cells were derived from the cerebellum of
neonatal mouse and immortalized by retrovirus-mediated v-myc
gene transfection [24,25]. They were routinely cultured to 70–
80% confluency in proliferation medium consisting of Dulbecco’s
Modified Eagle medium (DMEM, Invitrogen) supplemented with
10% fetal bovine serum (Invitrogen), and L-Glutamine (2 mM,
Invitrogen) in standard humidified incubators containing 5% CO2
For BrdU labeling, NSCs were seeded and plated onto
coverslips in 24-well plate (104 cells per well) in culture medium
consisting of Dulbecco’s Modified Eagle medium (DMEM,
Invitrogen) supplemented with 2%fetal bovine serum (Invitro-
gen), and L-Glutamine (2 mM, Invitrogen) in the presence of
LBP (1 mg/mL, 10 mg/mL) and/or 1 mM corticosterone (Cort).
After 44 h, BrdU (10 mM, Sigma) was added and the incubation
was performed for an additional 4 h. NSCs were fixed for
20 min with 4% paraformaldehyde, washed three times with
PBS (pH 7.2), and treated with 2 M HCl for 30 min at 37uC.
Cells were washed twice with PBS, blocked and permeabilized
in 10% normal goat serum plus 0.1% Triton X-100 for 1 h at
room temperature. After blocking, the cells were washed with
PBS and then incubated in primary rat-anti BrdU (1:200,
AbDSerotec) at 4uC overnight. After rinsing with PBS, the
coverslips were incubated with appropriate species-specific Alexa
Fluor 488-conjugated IgG (1:200, Invitrogen) antibody in the
dark at room temperature for 1 h. The coverslips were then
incubated in nuclei counterstained with DAPI (4,-6-Diamidino-
2-phenylindole, 1 mg/ml) and mounted in fluorescent mounting
medium (Dako). The fluorescence imaging was visualized by
using a Carl Zeiss Axio Observer Z1 fluorescent imaging
system. The numbers of total and BrdU-positive cells were
counted using microscopy in six non-overlapping fields per
coverslip. Results were expressed as relative percentage of
BrdU-positive cells. The images obtained were analyzed by
mean of Image J software (NIH, USA).
Figure 3. Corticosterone treatment inhibited sexual behavior, which is prevented by LBP treatment. (A): Intromission Latency; (B):
Copulatory Efficiency; (C): Ejaculation Frequency; (D): Ejaculation Latency. Data are presented as mean6SEM. *: p,0.05, ANOVA with LSD post-hoc
Wolfberry Improves Sexual Behavior
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Comparison among the different treatment groups were
conducted using One-way ANOVA, with LSD post-hoc test if
required. A statistically significant difference was indicated when
p#0.05. Data are expressed as mean 6 SEM.
LBP Facilitated Male Sexual Behavior
There were no significant differences in the ML, MF, IL and IF
found when comparing the three groups of rats treated with
different dosages of LBP (vehicle, 1 mg/kg and 10 mg/kg) at any
time-point (data not shown).
However, as shown in figure 2, ANOVA followed by post-hoc
test detected significant difference in the CE (Figure 2A), EF
(Figure 2B) and EL (Figure 2C) between the vehicle-treated and
LBP (1 mg/kg)-treated groups. The LBP (1 mg/kg)-treated
animals showed a significantly higher CE (Figure 2A) and EF
(Figure 2B) than vehicle-treated animals, while EL (Figure 2C) was
significantly decreased by the treatment (p,0.05 for all compar-
isons). The differences were found in all sexual behavior tests at
day 7, 14 and 21. In contrast, no difference was found when
comparing the LBP-treated group to vehicle group at dosage of
LBP Reversed Sexual Behavior Inhibition Induced by
Corticosterone resulted in decreased sexual performance when
compared to vehicles-treated control animals in terms of
significantly longer IL (Figure 3A), lower CE (Figure 3B), lower
EF (Figure 3C) and longer EL (Figure 3D, p,0.05 for the
comparisons). Co-treatment with LBP (1 mg/kg) significantly
reversed the inhibited sexual behavior, which is indicated by a
significantly lower IL (Figure 3A) and higher CE (Figure 3B) than
that of the corticosterone group (p,0.05). Although no significant
difference was found between the corticosterone group and co-
treatment group in EF and EL, the co-treatment group did not
show difference from the vehicle-treated group (Figure 3C and 3D).
LBP Reversed Suppressed Adult Neurogenesis Induced
In the SVZ, the number of BrdU-positive cells in the
corticosterone-treated animals was significantly lower than both
Figure 4. Effect of corticosterone and/or LBP treatment on SVZ and hippocampal neurogenesis. Representative microphotograph of
BrdU-positive cells in SVZ of (A) vehicle (B) corticosterone and (C) co-treatment with corticosterone and LBP groups are shown. Corticosterone
treatment significantly decreased cell proliferation when compared to vehicle (p,0.05) and co-treatment (p,0.01) groups (D). Representative BrdU-
staining of hippocampus are shown in (E [vehicle]), (F [corticosterone]) and (G [co-treatment]). Corticosterone group showed a lower number of BrdU-
positive cells than the vehicle group (H, p,0.05). Co-immunostaining of BrdU and DCX experiment (I-K) showed that co-treatment group showed a
significantly higher proportion of DCX/BrdU ratio than the corticosterone group (L) Arrows indicates co-labeled cells. Analyzed by ANOVA with LSD
post-hoc test. Data are expressed as mean6SEM. Scale bars: A-C & E-G: 50 mm; I-K: 25 mm.
Wolfberry Improves Sexual Behavior
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vehicle or co-treatment groups (Figure 4 A-D; p,0.05). Cortico-
sterone also decreased the number of BrdU-positive cells in the
hippocampus (Figure 2E-H, p,0.05). No difference was found
between the co-treatment group and vehicle group. The
proportion of DCX-expressing new cells (i.e. ratio of DCX/BrdU)
was significantly higher in the co-treatment group when compared
to vehicle or corticosterone groups (Figure 4I-L, p,0.05).
Corticosterone Suppressed Plasma Testosterone Level,
which is Prevented by LBP Treatment
Statistical analysis showed a significant decrease in plasma
testosterone level of corticosterone-treated animals (p=0.037
compared to PBS group). No significant difference could be found
between the PBS and Cort+LBP groups (Figure 5).
Adult Neurogenesis in SVZ and Hippocampus were
Correlated with Sexual Performance
When the sexual behavior data of the PBS, Cort and LBP+Cort
treated groups were plotted against the BrdU+ve cells in the SVZ
and hippocampus, significant correlations were found (Table 1).
Graphical representations of ML against BrdU positive cells in the
SVZ, IL against BrdU positive cells in the SVZ and IF against
BrdU positive cells in the hippocampus were shown in figure 6. In
general, high number of BrdU positive cells in both regions was
associated with a better sexual performance.
Blocking Neurogenesis Abolished the Pro-sexual Effect of
To determine the direct relationship between neurogenesis and
sexual behavior, neurogenesis in animals treated with LBP were
blocked with Ara-c infusion. Quantification of BrdU-positive cells
in the SVZ and hippocampus of control animals (SA group: saline
infusion and PBS feeding) and LBP/Ara-c treated animals (Ara-c
group) showed that cell proliferation was significantly inhibited in
the SVZ (SA: 618.3632.7; Ara-c: 74.5+2.34; p,0.01) and
hippocampus (SA: 8501.56551.6; Ara-c: 1561.7+283.8; p,0.01)
of Ara-c group. In contrast to experiment 1, blocking neurogenesis
in LBP-treated animals did not result in improvement of sexual
behavior in copulatory efficiency (Figure 7A), ejaculation frequen-
cy (Figure 7B) or ejaculation latency (Figure 7C).
LBP Increased Neural Stem Cell Line Proliferation and
Reversed the Suppressive Effect of Corticosterone in
In vitro study showed that LBP treatment at 1 mg/ml and
10 mg/ml increased cell proliferation in neural stem cell line C17.2
(Figure 8A & B, p,0.01). Similar to the results depicted in
experiment 2, corticosterone treatment suppressed the cell
proliferation of C17.2 cell line, while co-administration with
LBP at 10 mg/ml could reverse the suppression (Figure 8C & D,
The present study demonstrated the enhancing effect of LBP on
male sexual behavior in rats. In both experiments 1 and 2, the CE,
EF and EL were improved by LBP and only IL was also improved
in experiment 2. The effects of LBP appeared to enhance both
motivation (indicated by IL) and copulatory performance
(indicated by CE, EF and EL) . Furthermore, it is shown
that the improved sexual performance was associated with
increased neurogenesis in the two neurogenic zones, suggesting
that neurogenesis may be associated with the sexual-enhancing
effects of LBP. The causal relationship between neurogenesis and
sexual behavior was demonstrated in experiment 3, in which the
sexual-enhancing effect of LBP was abolished by blocking
Wolfberry has been prescribed for treating male infertility for a
long history, although there is a scarcity of scientific evidence for
this indication . Recently a study revealed that an improved
subjective view on sexual ability was found in subjects chronically
consumed a juice prepared from wolfberry . This study suggests
the potential clinical use of wolfberry to improve male sexual
The findings of the present study showed the sexual-enhancing
effect of LBP from a pre-clinical point of view. Our results showed
that the beneficial effect was not only revealed in normal, healthy
rats, but also in rats with sexual inhibition induced by
corticosterone. The ‘rescuing’ effect of LBP on male sexual
behavior suggests that LBP may be useful to treat male sexual
dysfunction. This notion agrees with previous studies in which
castration and irradiation on reproductive tissues suppressed male
mating behavior, while LBP improved the mating ability of the
affected rats [11,12]. Apart from behavioral aspect, LBP may exert
its beneficial effects on the reproductive system at cellular level.
For example, LBP was found to protect spermatogenic cells from
apoptosis induced by hyperthermia and the structural integrity of
Figure 5. Effect of corticosterone and/or LBP treatment on
plasma testosterone level in male rats. Data are represented by
mean6SEM. *: p,0.05.
Table 1. Correlational analysis of BrdU +ve cells in SVZ and
hippocampus and sexual behavior components.
Mount latencyr = 20.583; p,0.01
Intromission latencyr = 20.57; p,0.01
Intromission frequency r = 20.687; p,0.01
Copulatory efficiencyr =0.5; p=0.021
Ejaculation frequencyr =0.473; p=0.035
Ejaculation latencyr = 20.455; p=0.033
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the seminiferous epithelium could be preserved by LBP [10,11]. In
short, the sexual-enhancing effect of LBP may be multi-faceted,
ranging from protection of gametes against harmful insults to
enhancement of individual sexual behavior. Thus, how does LBP
benefit reproduction may warrant further investigations.
The relationship between neurogenesis and sexual behavior
shown in this study suggests that neurogenesis may be involved in
copulation, and neurogenesis could be one of the mechanisms of
how LBP exerts its pro-sexual effect. Neurogenesis could be found
at different brain regions while the two most prominent
neurogenic regions are the SVZ and hippocampus [27,28]. Neural
progenitor cells proliferate at the SVZ and migrate tangentially
along the rostral migratory stream (RMS) to reach the olfactory
bulb, which allows continuous addition of new neurons for
formation of new circuits throughout the adulthood in rodents
. The renewal of interneurons in the olfactory bulb renders the
ability of olfactory learning, including differentiating sex-related
olfactory signals . As the hippocampus is well recognized for its
necessity in memory formation, it may also take part in the
formation of memory regarding the processing of sexual cues .
Previous studies outlined the interrelationship between adult
neurogenesis and sexual behavior. For instance, exposure of
female prairie voles or ewes to males increased neurogenesis in the
Figure 6. Linear regression of ML and BrdU-positive cells in
SVZ (A), IL and BrdU-positive cells in SVZ (B) and IF and BrdU-
positive cells in hippocampus (C) in rats treated with PBS,
corticosterone or both corticosterone and LBP for 21 days.
Figure 7. Blocking neurogenesis abolished the pro-sexual
effect of LBP treatment. No significant difference between saline
infused-animals (SA) and LBP-treated animals with Ara-c infusion (Ara-c)
in (A) Copulatory Efficiency, (B) Ejaculation Frequency and (C)
Ejaculation Latency was found. Data are presented as mean+SEM. *:
p.0.05, Student’s t-test.
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SVZ and hippocampus [14,17]. Another study showed that
newborn cells in the olfactory bulbs integrated into the ‘mating’
circuit and could be activated by estrus female pheromones .
More recent studies showed that neurogenesis is essential for the
display of normal sexual behaviors in both female and male
rodents [13,18]. Under normal situation, female mice prefer to
mate with dominant males. When the neurogenesis in the SVZ
and hippocampus were blocked by cytostatic cytosine arabinoside,
the preference was then abolished . On the other hand, when
neurogenesis was suppressed in male rats, the sexual performance
was impaired . The decreased neurogenesis was associated
with a decrease in neuronal activation in mating-related circuits,
which may be the reason underlying the sexual inhibition. The
present study further showed that blocking neurogenesis could
abolish the sexual-enhancing effect of LBP. These observations
indicate that adult neurogenesis warrants the reproductive success
of rodents, and disruption of it would cause dysfunction in sexual
behavior. In the present data, corticosterone suppressed both
neurogenesis and sexual performance simultaneously while LBP
could prevent these from occurring. The neurogenesis promoting
effect of LBP was further confirmed in the in vitro assays, in which
the corticosterone-induced proliferation inhibition  was
reversed by LBP. Apart from cell proliferation, LBP also promotes
the neuronal differentiation of neural precursor cells as shown by
DCX staining. The neurogenic effect of LBP agrees with previous
findings . Interestingly, the sexual performance was found to
correlate with neurogenesis. These findings provide further
evidence to support the notion that the newborn neurons
participate in sexual behavior. Furthermore, neurogenesis in the
SVZ and hippocampus are correlated with different measurements
in the sexual behavior test, which implies that SVZ neurogenesis
may be intimately involved in sexual motivation (indicated by
ML/IL) while both the SVZ and hippocampus are involved in
copulatory performance. Further studies are required to explore
this possibility and this may provide insight on the neural basis of
Previous studies have shown a dose-dependent response of LBP
treatment on neuroprotection and sexual behavior . We
choose the current dosages (1 mg/kg and 10 mg/kg) since it was
reported that the maximum therapeutic effect could be achieved
by these two dosages [6,8,11]. Previous reports showed that high
dose of LBP inhibited c-jun N-terminal kinase (JNK) pathway
[20,35], which may subsequently inhibit neural stem cell
proliferation . This may be the potential mechanism of loss
of neurogenesis and sexual-enhancing effect of LBP at high
dosage, while this would be confirmed in future studies.
It should be noted that neurogenesis consists of different stages
including cell proliferation, differentiation, migration, survival and
incorporation of new neurons into the existing neural circuit .
The current study investigated the early phases including cell
proliferation and differentiation, while later phases were not
studied. While newly proliferated cells may act as a source of
trophic factors which affect functioning of other neurons, much
matured new neurons may directly involve in signal transduction.
Further studies of the effect of LBP on the later phases, which may
extend the treatment period to over eight weeks, may suggest its
long term effect on sexual behavior of the animal from the view of
LBP may also modulate sexual behavior via increasing the
expression of testosterone in sexually inhibited animals. Our
results show that chronic corticosterone significantly suppressed
the plasma level of testosterone; and thus resulting in inhibited
sexual performance. Concomitance to enhancing the sexual
performance, LBP also reverse the decrease in testosterone level.
Figure 8. LBP increased cell proliferation in C17.2 neural stem
cell line and reversed the suppressed neurogenesis by
corticosterone treatment. LBP treatment at 1 mg/ml and 10 mg/ml
increased cell proliferation when compared to control group (A & B).
While corticosterone (1 mM) suppressed cell proliferation, co-treatment
with LBP (10 mg/ml) reversed the suppression. Data are presented as
mean6SEM; **: p,0.01.
Wolfberry Improves Sexual Behavior
PLoS ONE | www.plosone.org8 April 2012 | Volume 7 | Issue 4 | e33374
Similar result was found by Luo et. al. [11,12] that LBP increased Download full-text
the testosterone levels in rats subjected to castration or irradiation
on testis. Alternatively, as it is recently reported that antioxidant
could increase the sexual motivation , LBP may enhance
sexual behavior through its antioxidant property .
To conclude, this study provides evidence for the pro-sexual
effect of LBP in both normal animals and animals with suppressed
sexual performance. Such findings support the use of wolfberry as
a treatment for sexual dysfunction or as an aphrodisiac treatment.
LBP could also reverse the decreased neurogenesis induced by
corticosterone, which is a possible mechanism underlying its effect.
Further exploration on the mechanisms would provide stronger
rationale for the use of wolfberry in clinical situations. Further-
more, the effect of wolfberry on female sexual function is not yet
explored. Future test of LBP on female rodent models may provide
insight on the potential usage of wolfberry on treating female
sexual dysfunction, such as sexual desire or arousal disorders.
The authors would like to thank Ms Jessica Yuen for her assistance with
Conceived and designed the experiments: BL YL RC KS. Performed the
experiments: BL JL YL YS SF. Analyzed the data: BL JL YL SF YS KS.
Contributed reagents/materials/analysis tools: JS RC KS. Wrote the
paper: BL JL YL RC KS.
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