Seasonal changes in expression of nerve growth factor and its receptors TrkA and p75 in the ovary of wild ground squirrel (Citellus dauricus Brandt)
Journal of Ovarian Research
Seasonal changes in expression of nerve growth factor and its receptors TrkA and p75 in the ovary of wild ground squirrel (Citellus dauricus Brandt)
Ben Li 0
Xia Sheng 0
Lihong Bao 2
Shiyang Huang 0
Qinglin Li 0
Yuning Liu 0
Yingying Han 0
Gen Watanabe 1
Kazuyoshi Taya 1
Qiang Weng 0
0 Laboratory of Animal Physiology, College of Biological Science and Technology, Beijing Forestry University , Beijing 100083 , China
1 Department of Veterinary Medicine, Faculty of Agriculture, Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology , Tokyo 183-8509 , Japan
2 Institute of Public Health, Inner Mongolia University for Nationalities , Tongliao 028000 , China
The aim of this study was to investigate the presence of nerve growth factor (NGF) and its receptors tyrosine kinase A (TrkA) and p75 in the ovaries of the wild ground squirrels during the breeding and nonbreeding seasons. In the breeding period, NGF, TrkA and p75 were immunolocalized in granulosa cells, thecal cells, interstitial cells and luteal cells whereas in the nonbreeding period, both of them were detected only in granulosa cells, thecal cells and interstitial cells. Stronger immunostaining of NGF, TrkA and p75 were observed in granulosa cells, thecal cells and interstitial cells in the breeding season compared to the nonbreeding season. Corresponding for the immunohistochemical results, immunoreactivities of NGF and its two receptors were greater in the ovaries of the breeding season then decreased to a relatively low level in the nonbreeding season. The mean mRNA levels of NGF, TrkA and p75 were significantly higher in the breeding season than in the nonbreeding season. In addition, plasma gonadotropins, estradiol-17 and progesterone concentrations were significantly higher in the breeding season than in the nonbreeding season, suggesting that the expression patterns of NGF, and TrkA and p75 were correlated with changes in plasma gonadotropins, estradiol-17 and progesterone concentrations. These results indicated that NGF and its receptors, TrkA and p75 may be involved in the regulation of seasonal changes in the ovarian functions of the wild ground squirrel.
Ground squirrel; NGF; Ovary; p75; TrkA
The nerve growth factor (NGF) belongs to a family of
related proteins required for the survival, maintenance, and
development of discrete neuronal populations in the central
and peripheral nervous systems [1-3]. It is also believed that
NGF not only has an effect on the nervous system, but also
plays an important role in a variety of non-neuronal system,
such as immune, cardiovascular and endocrine systems
[4-6]. The effect of NGF has been shown to be mediated
through specific membrane receptors high-affinity tyrosine
kinase A (TrkA), which is responsible for its biological
activities [7,8]. Furthermore, the effect of NGF is also
mediated via low affinity receptor p75 that also functions as
other neurotropins receptor . When p75 and TrkA
receptors are co-expressed, p75 increases the sensitivity of
the TrkA receptor and its signaling efficiency [10,11].
It is now well known that NGF and its receptors are
expressed in the mammalian ovary, including women
[12-14], rats [14,15], golden hamsters [16-18], cows ,
sheep  and Shiba goats . More and more
evidences have indicated that NGF signaling plays a critical
role in the development of mammalian ovary, oogenesis
and folliculogensis [22-24], in an auto- and/or paracrine
manner. In our previous studies of the golden hamsters,
NGF and its two receptors TrkA and p75 were present in
ovaries, oviducts and uteri, demonstrating that NGF, TrkA
and p75 have important autocrine and paracrine regulatory
roles in the function of reproductive organs during the
estrous cycle [16,25,26]. Data to support this concept in
wild animals, however, is very limited. To study the basic
mechanisms of NGF regulation of ovarian function during
the breeding and nonbreeding seasons within the annual
reproductive cycle, the wild ground squirrel offers a useful
model without any manipulations.
The wild ground squirrel (Citellus dauricus Brandt) is a
typical seasonal breeder which has a strict and extremely
compressed breeding period (for female individuals, it
includes estrous period, pregnancy and birth) from April
to May and a long period of sexual dormancy from June
to the following March including a 6-month hibernation
period . The wild female ground squirrel exhibits
estrus immediately after emergence from hibernation in
spring, and has a gestation period of 28 days . Whether
fertilized or not, all females become sexually inactive as the
relatively brief breeding season ends. Previously, we
observed the presence of inhibin/activin subunits in the
ovary of the wild ground squirrels, indicating its important
paracrine/autocrine regulatory role in the seasonal
folliculogenesis of the wild ground squirrel . In search for
other key local players, we investigated the expression
levels and immunolocalization of NGF and its receptors,
TrkA and p75 in ovarian tissues of the wild ground
squirrel during the breeding and nonbreeding seasons, and to
elucidate the relationship between NGF and its receptors
(TrkA and p75) and ovarian functions in this wild rodent.
Material and methods
All the procedures on animals were carried out in
accordance with the Policy on the Care and Use of Animals by
the Ethical Committee, Beijing Forestry University and
approved by the Department of Agriculture of Hebei
province, PR China (JNZF11/2007). Wild female ground
squirrels that were regarded as adults according to their
body weights (242412 g) were captured on April 13 (10.2
hours of daylight) after emergence from hibernation in
the breeding period (n = 10) and on June 9 (12.6 hours of
daylight) in the nonbreeding period (n = 8) of 2009 in Hebei
Province, PR China.
Animals were anesthetized with 4% isoflurane and blood
samples were rapidly collected from leg vein. Plasma
samples were frozen and stored at -20C, after the blood
samples were added heparin sodium and centrifuged
(3000 rpm, 20 min at 4C). An overdose of pentobarbital
was applied afterwards for euthanasia. Ovary and brain
were quickly removed and dissected. Length, width and
weight of each ovary were measured. A part of the tissues
were fixed in 0.05 M phosphate-buffered saline (PBS,
pH 7.4) containing 4% paraformaldehyde for histological
and immunohistochemical observation, while the rest were
immediately frozen in liquid nitrogen and stored at -80C
for RNA isolation and protein extraction.
Ovarian samples were dehydrated in ethanol series and
embedded in paraffin wax. Serial sections (4 m) were
mounted on slides coated with poly-L-lysine (Sigma,
St. Louis, MO, U.S.A.). Sections were stained with
hematoxylin-eosin (HE) for observations of general
histology. The sections were screened using an Olympus
photomicroscope with a 20 objective lens and imaged
with software Image-Pro Plus 4.5 (Media Cybernetics,
Bethesda, MD, USA). Every one in ten serial sections,
and altogether 50 and 30 sections for the breeding and
nonbreeding season ovary respectively were selected for
follicle identification  and quantification. Six random
vision fields were selected per Section.
Ovarian sections were blocked with 10% normal goat
serum to prevent the non-specific binding of the second
antibody. The sections were then incubated with polyclonal
primary antibody against NGF (0.4 g/ml, M-20), TrkA
(2 g/ml, 763) or p75 (2 g/ml, H-92) (Santa Cruz
Biotechnology, Santa Cruz, CA, USA) for 12 h at 4C, and
incubated with the second antibody, goat anti-rabbit IgG
conjugated with biotin and peroxidase with avidin for 1 h
at room temperature. The sections were visualized using a
rabbit ExtrAvidin staining kit (Sigma, St. Louis, MO, USA)
in 150 ml of 0.05 M TrisHCl buffer containing 30 mg
3,3diaminobenzidine (Wako, Tokyo, Japan) plus 30 l H2O2.
Finally, the sections were counterstained with hematoxylin
(Merck, Tokyo, Japan) and NGF, TrkA and p75 were
detected, respectively. The immunostained slides were
scanned using the software Image-Pro Plus 4.5 (Media
Cybernetics, MD, USA) at 20 magnification. The specificity
of NGF and its receptors, TrkA and p75 antibodies have
been described previously . The immunohistochemical
staining was determined as positive (+), strong positive
(++), very strong positive (+++), and negative (). Staining
that was weak but higher than control was set as positive
(+); the highest intensity staining was set as very strong
positive (+++); staining intensity between + and +++ was
set as strong positive (++).
Ovarian tissues were weighed and dissected into small
pieces using a clean razor blade. The tissues were
homogenized in a tissue homogenizer containing 300 l
of 10 mg/ml PMSF and incubated for 30 min on ice.
Homogenates were centrifuged at 12,000 g for 10 min at
4C. Protein extracts (25 g) were mixed with equal
volumes of 2 Laemmli sample buffer. Equal amounts of
proteins from each sample were loaded onto a 12%
SDSPAGE gel and electrophoretically separated at 18 V/cm
and transferred to nitrocellulose membrane using a wet
transblotting apparatus (Bio-Rad, Richmond, CA, USA).
The membrane was blocked in 3% BSA for 1 h at room
temperature. Primary incubation of the membrane was
carried out using NGF, TrkA or p75 antibody (1:1000
dilution) for 1 h at room temperature. Secondary
incubation of the membrane was then carried out using an IRDye
(1:5000 dilution, Rockland, Gilbertsville, PA, USA) for 1 h
at room temperature. Finally, the membrane was washed
in 25 ml Tris-Buffered Saline with Tween-20 (TBST wash
buffer, 0.02 M Tris, 0.137 M NaCl and 0.1% Tween-20,
pH 7.6) plus 3 l H2O2 and visualized with Odyssey
infrared imaging system. Brain tissue of wild ground squirrel
was used as a positive control and water, instead of
primary antisera, was used as a negative control. -actin was
selected as the endogenous control. The intensities of the
bands were quantified using Quantity One software
(Version 4.5, Bio-Rad Laboratories) and expression ratios were
Total RNA from each sample was extracted using
ISOGEN (Nippon Gene, Toyama, Japan). Approximately 1 g
of ovarian tissues were thawed and immediately
homogenized in 10 ml of ISOGEN. The homogenate was
incubated for 5 min at room temperature to allow the
complete dissociation of nucleoprotein complexes. After
the addition of 2 ml of chloroform, the mixture was
vigorously shaken for 3 min at room temperature and
centrifuged at 12,000 g for 10 min at 4C. The aqueous
phase was then transferred to a fresh tube and washed
with an equal volume of chloroform. An equal volume of
isopropanol was added, and the sample was kept for
10 min at room temperature. RNA was precipitated by
centrifugation at 12,000 g for 10 min at 4C. The RNA
pellet was washed twice with 75% ethanol, briefly dried
under air, and dissolved in 100 l of
Reverse transcription-polymerase chain reaction (RT-PCR)
The first-strand cDNA from total RNA was synthesized
using Superscript II Reverse Transcriptase (Invitrogen,
Carlsbad, CA, USA) and oligo (dT)1218 according to
the manufacturers protocol. The 20 l of reaction
mixture contained 4 g of total RNA, 0.5 g of oligo
(dT)1218, 2.5 mM MgCl2, 0.5 mM dNTP, 10 mM
dithiothreitol, 20 mM TrisHCl (pH 8.4) and 200 U of
Superscript II enzyme. The first-strand cDNA was used
for PCR amplification with the appropriate primers
previously proved (Table 1). Given the unknown
CAAGAGACCGAGCAATCAAG Page 3 of 9
genome of the wild ground squirrel, we could only
design primers based on the sequence of mouse and
rat , considering the relative conserved sequences
between these rodents. The 100 l of reaction mixture
contained 1 l of first-strand cDNA, 0.5 M each
primer, 1.5 mM MgCl2, 0.2 mM dNTP, 20 mM
TrisHCl (pH 8.4) and 2.5 U of Taq polymerase
(Invitrogen, Carlsbad, CA, USA). The amplification
was under the following condition: 94C for 5 min for
the initial denaturation of the RNA/cDNA hybrid, 30 cycles
of 94C for 1 min, 52C for 1 min, and 72C for 2 min for
amplification. The PCR product was electrophoresed in the
2% agarose gel and individual bands were visualized by
ethidium bromide staining. Brain tissue of wild ground squirrel
was used as positive control and water, instead of cDNA,
was used as negative control. The housekeeping gene,
RpL7, was selected as the endogenous control as it is an
estrogen-independent gene. The bands were quantified
using Quantity One software (Version 4.5, Bio-Rad
Laboratories) and expression ratios were calculated.
Cloning and sequencing of PCR products
The purified PCR products were ligated into pCR
2.1TOPO (Invitrogen, Carlsbad, CA, USA) and the ligation
products were used to transform the competent E. coli
using TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA,
USA). Plasmids were extracted from the bacteria and
positive clones containing the proper insert were sequenced in
both directions using Thermo Sequenase II Dye
Terminator Cycle Sequencing Premix Kit (Amershan Pharmacia
Biotech, UK) with an automatic sequencing system (ABI
PRISM 377, Applied Biosystems Japan, Tokyo, Japan).
After obtaining the sequence of each PCR product, we
blasted with the known mRNA sequences of mouse
(NGF, NM_013609.3; TrkA, NM_001033124.1; p75,
NM_033217.3; RpL7, NM_011291.5), rat (NGF, NM_0012
77055.1; TrkA, NM_021589.1; p75, NM_012610.2; RpL7,
NM_001100534.1), bovine (NGF, NM_001099362.1; TrkA,
XM_005898751.1; p75, NM_001102478.2; RpL7, NM_001
014928.1) and human (NGF, NM_002506.2; TrkA, AB0194
88.2; p75, NM_002507.3; RpL7, NM_000971.3), find the
homologous sequence fragments in each species and
compare for homology using DNAman.
Plasma concentrations of estradiol-17 and
progesterone were determined by double-antibody RIA systems
using 125I-labeled radioligands as described previously
. Antisera against estradiol-17 (GDN 244) 
and progesterone (GDN 337)  was kindly provided
by Dr. G. D. Niswender (Animal Reproduction and
Biotechnology, Colorado State University, Fort Collins,
CO). The intra- and inter-assay coefficients of
variation were 3.7% and 6.2% for estradiol-17 and 6.3%
and 15.4% for progesterone, respectively. Plasma
concentrations of follicle stimulating hormone (FSH) and
luteinizing hormone (LH) were measured by
doubleantibody RIA systems using a rabbit antiserum against
human FSH (#6; provided by NIDDK NIH, Bethesda,
MD, USA) and a rabbit antiserum against ovine LH
(YM #18; provided by Dr Y. Mori, Laboratory of
Veterinary Ethology, University of Tokyo, Tokyo, Japan).
The intra- and inter-assay coefficients of variation
were 9.2% and 13.2% for FSH and 8.8% and 13.0% for
Means and standard deviations were calculated. Data were
analyzed using a one-way ANOVA and the means were
compared for significance using Duncans Multiple Range
Test (P = 0.05) using the SPSS computer software package.
The distinct variation in ovarian histology and
Ovaries of breeding and nonbreeding seasons were
observed morphologically and histologically (Figure 1).
In line with our previous reports, both the ovarian
volume and weight were markedly higher in the
breeding season than in the nonbreeding season (Figure 1a
and b, p < 0.01). All levels of follicles, as well as the
corpora lutea, were seen in the breeding season ovary
(Figure 1c), whereas primary and secondary follicles
comprised most of the nonbreeding season ovary, with
few tertiary and mature follicles (Figure 1d). Based on
the HE staining of the serial sections, we also
quantified the numbers of different levels of follicles and
corpora lutea (Figure 1e and f ). Apparently, the lower
ratio of primary follicles and higher ratios of
secondary, tertiary, mature follicles and corpora lutea implied
a more active follicuologenesis in the breeding period
ovary. The distinct folliculogenesis of the breeding and
nonbreeding seasons were schemed respectively in
Figure 1g and h.
The ovarian immunoreactivity of NGF, TrkA and p75
Immunohistochemistry was performed to detect the
localization pattern of NGF and its receptors in the wild
ground squirrel ovary and representative stainings were
shown in Figure 2. In the breeding season, both NGF
ligand and TrkA receptor were present in various types
of cells, including the granulosa cells, theca cells,
interstitial cells and corpora lutea, with the highest intensity
in the granulosa cells (Figure 2b,c,f,g). Strong positive
signals of p75 were also detected in these somatic
cells, but not in the corpora lutea (Figure 2d and h).
The immunostaining intensity generally decreased when
it came to the nonbreeding season. NGF and TrkA were
still positively stained in the secondary follicles of the
nonbreeding season ovary, where mild signals of p75
were detected only in the granulosa and theca cells
(Figure 2j,k,l). No signal was seen in the negative
control panel (Figure 2a,e,i). The immunoreactivity of each
staining was quantified and summarized in Table 2.
The seasonal changes in ovarian protein and mRNA
expressions of NGF, TrkA and p75
We then moved on to detect the protein and mRNA
expression levels of NGF, TrkA and p75 using Western
Figure 1 Morphological and histological features of ovarian tissues of the wild ground squirrel during the breeding and nonbreeding
periods. Marked seasonal differences were observed in ovarian volume (a) and weight (b). Subsequently, HE staining was performed for the
ovaries of the breeding season (c) and nonbreeding season (d), and follicles of the two periods were manually quantified accordingly (e and f).
g and h were the schematic diagrams for the varied folliculogenesis of the breeding and nonbreeding periods. B, breeding season; NB,
nonbreeding season; MF, mature follicle; CL, corpus luteum; SF, secondary follicle. *, P < 0.01.
Figure 2 Immunohistochemical localization of NGF ligand and receptors in ovaries of the wild ground squirrel during breeding and
nonbreeding periods. The first column (a, e, i) represents negative control samples of the breeding season ovary, corpus luteum and
nonbreeding season ovary; the second column (b, f, j), the third column (c, g, k), and the fourth column (d, h, l) represent immunostaining with
antibodies against NGF, TrkA and p75, in the breeding period ovary, corpus luteum and nonbreeding period ovary. B, breeding period; NB, the
nonbreeding period; GC, granulosa cells; TC, theca cells; IC, interstitial cells; LC, luteal cells. Scale bar, 50 m.
blot and PCR, and representative bands were shown in To further confirm the nature of the PCR signals, cDNA
Figure 3. The intensity of each band was normalized to fragments of NGF, TrkA and p75 in ovarian tissues were
the level of -actin and RpL7, used as the endogenous sequenced and compared to the corresponding fragments
control, for Western and PCR detections respectively. in mouse, rat, bovine, and human. The partial mRNA
Both the protein and mRNA expression levels of NGF sequences in the wild ground squirrel are as below:
ligand and receptors were significantly higher in the
breeding season when compared to the nonbreeding season,
basically in consistent with the immunohistochemical
results (Figure 3, p < 0.01). Notably, the relative protein level
of NGF and the relative mRNA levels of NGF, TrkA and
p75 were even higher in the breeding season ovary than in
the brain (P < 0.05), reassuring the substantial role of NGF
during this particular time.
Table 2 Relative abundance of NGF, TrkA, and p75 in
ovaries of the wild ground squirrels during the breeding
and nonbreeding seasons
Immunoreactivity was shown as - for negative staining, + for the positive
staining, ++ for the strong positive staining, +++ for the very strong positive
staining and/for no such cell type.
Figure 3 Western blot and RT-PCR detections of NGF, TrkA and p75 in ovarian tissues of the wild ground squirrel during the breeding period
(B) and nonbreeding period (NB), respectively. The left column shows the western bolt results of NGF (A), TrkA (B) and p75 (C) respectively, The right
column shows the RT-PCR results of NGF (D), TrkA (E) and p75 (F) respectively. The proteins extracted from brains were used as the positive control (PC).
Water was used as the negative control (NC). -actin blots shown were used as controls to correct for loading in each lane. The expression levels were
determined by densitometric analysis. Bars represent means + SD for five independent experiments. Means within the columns marked with different
letters indicate significant difference (P < 0.01).
GACGATGCCGTGTGCCGATGCTCCTATGGCTAC The 176-bp NGF cDNA nucleotide sequence identity
TACCAGGACGAGGAGACTGGCCGCTGCGAGGC was 92.11%, 90.20%, 81.70% and 86.18%, respectively;
TTGCAGCGTGTGCGGGGTGGGCTCAGGATCGG the 261-bp TrkA cDNA nucleotide sequence identity
TGTCCTCCC was 94.32%, 90.83%, 84.72% and 83.41%, respectively; the
RpL7# 295-bp p75 cDNA nucleotide sequence identity was
AAGGATCTGCTGCTGCTTCTGTTCCAGATCTCA 94.42%, 88.10%, 84.01% and 83.64%, respectively; the
ATGGCACCTTTGTTAAGCTCAACAAGGCTTCAA 207-bp RpL7 cDNA nucleotide sequence identity was
TTAACATGCTGCGGATTGTGGAGCCATACATTG 94.55%, 84.65%, 74.23% and 76.87%, respectively (Table 3),
CATGGGGGTACCCCAACCTGAAGTCAGTAAACG which not only confirmed the specificity of PCR primers
AGCTCATCTACAAGCGAGGCTACGGCAAAATCA but also suggested high affinities of NGF, TrkA and p75
ACAAGAAGCGGATTGCCTTGACAGATAATTCCT genes between the wild ground squirrel and the species
Table 3 Nucleotide sequence identity in ovarian tissues
of wild ground squirrel in comparison with mouse, rat,
bovine and human (%)
Plasma concentration of LH, FSH, estradiol-17 and
The profiles of plasma LH, FSH, estradiol-17 and
progesterone are shown in Figure 4. Plasma LH, FSH,
estradiol-17 and progesterone concentrations were all
remarkably higher in the breeding season as compared
to the nonbreeding period (Figure 4a,b,c,d, p < 0.01).
The present study demonstrated that
immunoreactivities of NGF and its two receptors, TrkA and p75 were
greater in the ovaries of the breeding season then
decreased to a relatively low level in the nonbreeding
season, and the expression patterns of NGF ligand
and receptors were correlated with the changes of
plasma concentrations of gonadotropins, estradiol-17
and progesterone. These findings suggested that NGF,
TrkA and p75 may be involved in the regulation of
seasonal changes in the ovarian functions of the wild
The present histological result was in agreement with
previous data reported in this species, that the number
of primary follicles had no significant difference
Figure 4 Seasonal change of the plasma concentration of LH,
FSH, estradiol-17 and progesterone. Plasma LH (a), FSH (b),
estradiol-17 (c), and progesterone (d) levels in the wild ground
squirrel during breeding period (B) and nonbreeding period (NB).
Bars represent means + SD for five independent experiments. Means
within the columns marked with different letters indicate significant
difference (P < 0.01).
between the breeding and nonbreeding seasons,
whereas the number of secondary follicles, antral follicles,
post-antral follicles and corpus luteum displayed a
significantly decrease from the breeding season to the
nonbreeding season [28,29]. It suggested that primary follicles
were stopped from developing to the stage of secondary
follicles, antral follicles, post-antral follicles during
nonbreeding season in the wild ground squirrel ovary. The
fact that corpus luteum was not discovered in the
nonbreeding season strongly suggested that no ovulation
occurred in this stage . In the present study, NGF
and its receptors, TrkA and p75 were immunolocalized
in ovarian tissues during the breeding and nonbreeding
period, and both mRNA and protein of NGF, TrkA and
p75 were also detected, indicating in situ synthesis and
secretion of NGF and its receptors in ovaries of the wild
ground squirrel, where the role of NGF might be
mediated via both receptors to affect follicular development.
Moreover, the expression levels of NGF and its
receptors, TrkA and p75 were significantly higher in the
breeding season as compared to the nonbreeding
season, implying that NGF system may be involved in the
regulation of ovarian function change. Our findings in
the wild ground squirrel was generally in line with
previous reports in pig and ewe, which showed higher
contents of NGF and TrkA in large follicles than in smaller
follicles [20,35]. Given the increased expression of NGF
and its receptors in ovary of the breeding season, as
well as the distinct morphological change, it is
reasonable to postulate that NGF contributes to not only the
innervation of rapidly growing follicles, but also
steroidogenic cell proliferation and steroid production in
the breeding season ovary, as has been observed in
other rodents [16,36,37].
The female reproductive system undergoes a number
of programmed cyclical processes during the course of
the ovulatory cycle. NGF and its receptors, under the
influence of gonadotropins and/or ovarian hormones, may
play a crucial regulatory role in these processes . In
the present study, the expression of NGF and its
receptors were correlated with changes in plasma
concentrations of LH, FSH, estradiol-17 and progesterone during
the breeding and nonbreeding seasons. These results
were similar to those found in golden hamsters and pigs.
Previous studies in golden hamster have suggested that
LH surge may be an important factor for inducing the
expression of NGF, TrkA, p75 in ovarian tissues
periodically [16,18]. In pig ovary, the increase in expression of
NGF and TrkA in large follicles found on day 20 may also
result from an LH effect . Dissen et al. reported that in
juvenile rats treated with equine chorionic gonadotropin,
significantly elevated TrkA mRNA levels were found in
ovary after the first preovulatory LH surge . Similarly,
the functional relationship between gonadotropins and
NGF in studies of the sheep ovary showed that large
follicles respond with increased NGF release to in vitro
stimulation with a combination of LH and FSH . Earlier
studies also indicated that NGF activity and content were
found to be increased by estradiol in a glioma cell line
culture . In addition, our previous studies showed that the
expression of NGF, TrkA and p75 in the uterus of the wild
ground squirrel was highest in the breeding season, when
estradiol and progesterone production were greatest .
Taken together, the present results are in accordance with
the views that NGF and its receptors are expressed in
ovarian tissues, and these expressions are gonadotropins
and/or ovarian hormones-dependent, and therefore, may
contribute to events either led to or associated with the
ovulatory process .
The present study showed that immunolocalization
for NGF and its receptors were also found in luteal cells
during the breeding season, which was similar to those
observed in other species such as golden hamster ,
Shiba goat  and porcine . In golden hamster,
luteal cells displayed a stronger reaction for NGF and its
receptors in metestrus than in estrus and diestrus .
In pig, the increased NGF and TrkA protein levels in
luteal cells were found during the estrous cycle .
These results shed light to the critical role of NGF in
development and/or maintenance of luteal function,
which is further supported by the findings that NGF can
stimulate of progesterone and oxytocin release  and
acetylcholine production  in bovine luteal cells as
well as maintenance of luteal vasculature in rats . In
line with these aforementioned data, our present results
indicated that NGF ligand and receptors might play a
similar role in the development of corpora lutea in the
wild seasonally-breeding rodent.
Cellular growth is related to the ability to promote
proliferation of mesenchyme and follicular cells, as well as to
induce FSHR synthesis [13,15]. In our previous studies,
the Western blotting results of FSHR showed that
significant induction in the breeding season compared with the
nonbreeding season . This study revealed that the
change levels of gonadotropins were parallel to those in
the expression patterns of ovarian NGF and receptors
during the breeding and the nonbreeding seasons. In rats,
ovaries treated with NGF developed the capacity to
response to FSH, with the formation of cAMP in preantral
follicles . Similar results were obtained in human
cells, in which the culture of granulosa cells with NGF
also increased expression of the FSHR in these cells .
These results implied that NGF may act indirectly in
follicular development through the production of
biologically active FSHR . In golden hamsters, as one
member of TGF superfamily, inhibin -subunit might
works in concert with NGF and its receptors to act on
LH/hCG receptor expression in ovarian interstitial cells
and associated with the ovulatory process . Also, our
previous studies showed that inhibin/activin subunits
(, (A) and (B)) were present in granulosa cells, theca
cells of antral follicles and interstitial cells in the breeding
season ovary of wild ground squirrel, following ovulation,
the corpora lutea become a major site of inhibin/activin
synthesis . Moreover, the expression patterns of
inhibin/activin subunits in the wild ground squirrel ovary
were also consistent with the present results. Thus,
combined with previous reports, this study suggested that the
co-expression of NGF and its receptors, along with other
key growth factors, including inhibin/activin subunits,
might indicate synergistic actions of them in the regulation
of seasonal folliculogenesis in the wild ground squirrel.
In conclusion, we have shown, for the first time, the
expression patterns of ovarian NGF, TrkA and p75
during the breeding and nonbreeding seasons in the wild
ground squirrel. Our results revealed a close correlation
between NGF expression and gonadotropins and steroid
hormones, which implicated that NGF ligand and
receptors are likely to be involved in the regulation of
seasonal changes in the ovarian functions of the wild
ground squirrel. The interesting alterations in ovarian
morphology and histology as well as folliculogenesis
observed between breeding and nonbreeding periods in
the wild ground squirrel demonstrate a complex
regulatory mechanism (s) that involves both sex steroids and
BL participated in performing the experiments, analyzing the data and
drafting the manuscript. XS, LB, SH, QL and YL assisted with sample
collection, all experiments and helped revising the manuscript. YH and QW
designed, supervised the study, and revised manuscript. GW and KT
provided some reagents and revised the manuscript. All authors read and
approved the final manuscript.
This study is supported by a Grant-in-Aid from the Program for the
Fundamental Research Funds for the Central Universities (BLYX2013024)
and National Natural Science Foundation of China (NSFC, No. J1103516)
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