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Int J Pharm Biomed Res 2012, 3(3), 141-146
Research article
International Journal <strong>of</strong>
PHARMACEUTICAL
AND BIOMEDICAL
RESEARCH
ISSN No: 0976-0350
<strong>Effect</strong> <strong>of</strong> <strong>ghrelin</strong> <strong>hormone</strong> on ovary histology in female wistar albino rats
S. Antony Selvi*, D. Raja Kumar, K. Rekha
Department <strong>of</strong> Physiology, Rajah Muthiah Medical College, Annamalai University, Chidambaram-608002, Tamil Nadu, India.
Received: 08 Aug 2012 / Revised: 13 Aug 2012 / Accepted: 14 Aug 2012 / Online publication: 16 Aug 2012
ABSTRACT
Aim: The aim <strong>of</strong> our present study is to assess the histophysiological changes in the ovary <strong>of</strong> immature and mature
wistar strain female albino rats in response to the intraperitoneal injection <strong>of</strong> <strong>ghrelin</strong> <strong>hormone</strong>. Methods: Eighteen immature
female albino rats (65 - 70 g) and eighteen mature female albino rats (130 to 140 g) were randomly allocated into control and
treatment groups (two doses as low dose - 10µg/kg and optimum dose - 20µg/kg <strong>of</strong> body weight <strong>of</strong> <strong>ghrelin</strong>).Treatment groups
were injected with <strong>ghrelin</strong> for 15 days .On 16 th day the animals were sacrificed. The body weight and weight <strong>of</strong> the ovaries
were measured. The ovaries were fixed in10 % buffered formalin and stained with haematoxylin and eosin. The histoarchitecture
<strong>of</strong> ovary was studied. Results: Administration <strong>of</strong> <strong>ghrelin</strong> indicates well defined changes like reduced number <strong>of</strong>
graffian follicles, some <strong>of</strong> which show mild degenerative changes like disorganized cells. Reduction in number <strong>of</strong> corpus
luteal cells, prominent Stromal tissues, congestion <strong>of</strong> blood vessels were also seen in the ovaries <strong>of</strong> animals in mature female
rats <strong>of</strong> optimum dose. There was no significant change in histoarchitecture <strong>of</strong> immature ovary <strong>of</strong> treated groups. In immature
and mature group there was significant increase in body weight but decreased ovarian weight in treated animals when
compared with control. Conclusion: From the present study it may be concluded that <strong>ghrelin</strong> produced degenerative changes
in the ovaries <strong>of</strong> mature albino rats and thus it may have a negative influence on reproductive function and affects fertility.
Key words: Female albino rats, Fertility, Folliculogenesis, Ghrelin harmone
1. INTRODUCTION
Ghrelin is a 28-amino acid peptide, derived from
prepro<strong>ghrelin</strong>. Ghrelin was identified as an endogenous
ligand for growth <strong>hormone</strong> secretagogue receptor (GHS-R)
from the stomach in 1999 [1]. It has two major endogenous
forms: a des-acylated form (des-acyl <strong>ghrelin</strong>) and a form
acylated at serine 3 position (<strong>ghrelin</strong>). This post translational
acylation is essential for the <strong>hormone</strong> biological activity
[1-3]. Ghrelin and its receptor – growth <strong>hormone</strong>
secretagogue receptor (GHS-R1a) were expressed in the key
cells <strong>of</strong> both male and female reproductive organs in several
species. The expression <strong>of</strong> <strong>ghrelin</strong> has also been reported in
pancreas, lymphocytes, kidney, lung, heart, pituitary, brain,
ovary, testis and placenta [4-6].
*Corresponding Author. Tel: +91 9442360859 Fax: +91 4144 238499
Email: antony.selvi@yahoo.com
©2012 PharmSciDirect Publications. All rights reserved.
The majority <strong>of</strong> circulating <strong>ghrelin</strong> is synthesized and
secreted by X/A-like cells localized within the oxyntic glands
<strong>of</strong> the mucosa <strong>of</strong> the gastric fundus [7].The hypothalamus has
been identified as the main source <strong>of</strong> <strong>ghrelin</strong> in the central
nervous system. In rats, systemic administration <strong>of</strong> <strong>ghrelin</strong>
reduces in vivo the GnRH pulse frequency. The involvement
<strong>of</strong> NPY in the mediation <strong>of</strong> the effects <strong>of</strong> <strong>ghrelin</strong> on pulsatile
GnRH secretion is indicated by the complete abolition <strong>of</strong> the
effects <strong>of</strong> <strong>ghrelin</strong> by the NPY-Y5 receptor antagonist [8].
GnRH secretion by hypothalamic fragments from
ovariectomized females is also significantly inhibited by
<strong>ghrelin</strong> [9]. It was found that both intracerebroventricular and
intraperitoneal administration <strong>of</strong> <strong>ghrelin</strong> in freely feeding rats
stimulated food intake [10].
Emerging evidence strongly indicates that the <strong>ghrelin</strong> and
<strong>ghrelin</strong> receptors (GHS-R1a and GHS-R1b) are present in the
mammalian and nonmammalian ovary. For example, <strong>ghrelin</strong>
is found in human, rat, pig, sheep, and chicken ovary
[11–15]. More precisely, in rodent ovary, expression <strong>of</strong>
<strong>ghrelin</strong> has been demonstrated in steroidogenically active

S. Antony Selvi et al, Int J Pharm Biomed Res 2012, 3(3), 141-146 142
luteal and interstitial hilus cells. These observations indicate
that ovarian follicular and luteal cells are potential targets for
systemic or locally produced or exogenously induced <strong>ghrelin</strong>,
because they express the functional type 1a <strong>of</strong> GHS-R. It also
emphasizes the plausibility for a role <strong>of</strong> <strong>ghrelin</strong> in the direct
control <strong>of</strong> ovarian cell functions.
In mammalian and nonmammalian species, <strong>ghrelin</strong> affects
gonadotropin release acting at the level <strong>of</strong> the hypothalamus
as well as directly on the pituitary gland [16]. In pituitary,
<strong>ghrelin</strong> suppresses LH pulse frequency in rats, sheep,
monkeys [17-19] and humans [20]. In rats, <strong>ghrelin</strong> is able to
down regulate Kiss1 expression in the hypothalamic medial
preoptic area and this could be a contributing factor in
<strong>ghrelin</strong>-related suppression <strong>of</strong> pulsatile LH secretion [21].
There is evidence in rats and human that <strong>ghrelin</strong> can suppress
not only LH but also FSH secretion in males and females
[19].These findings contribute an impact that <strong>ghrelin</strong> not only
affects ovulation but also folliculogenesis in rats.
A mounting body <strong>of</strong> evidence indicates that <strong>ghrelin</strong>
participates in the regulation <strong>of</strong> reproductive physiology by
two actions:
(i) Through systemic release <strong>of</strong> the stomach- derived peptide,
which acts at different levels <strong>of</strong> the reproductive system.
(ii) Through biological actions on reproductive organs by
locally expressed <strong>ghrelin</strong> [4,22,23]. As previously
mentioned, it has been suggested that <strong>ghrelin</strong> has a role in
the control <strong>of</strong> reproduction. This is because <strong>of</strong> the effects
<strong>of</strong> locally produced <strong>ghrelin</strong> as well as the gut derived
<strong>hormone</strong> conferring systemic control <strong>of</strong> reproduction and
the direct gonadal effects [22,24-26].
On the basis <strong>of</strong> the above cited references, to ovary
<strong>ghrelin</strong> expression and its inhibitory effect on ovarian cells, it
is tempting to have a study on histophysiological effect <strong>of</strong>
intraperiotneal administration <strong>of</strong> <strong>ghrelin</strong> and to hypothesize
that <strong>ghrelin</strong> might play a role in the control <strong>of</strong> reproductive
function and may have a negative influence on fertility.
2. MATERIALS AND METHODS
This study was conducted in the Department <strong>of</strong>
Physiology, Rajah Muthiah Medical College, (RMMC),
Annamalai University, Chidambaram, Tamil nadu, India.
2.1. Materials
Ghrelin, rat with n-octanoyl modification at Serine 3, was
obtained from anaspec chemicals (catalog number: 24159)
2.2. Selection <strong>of</strong> animals
Immature and mature female albino rats <strong>of</strong> wistar strain
weighing 65 to 70g and 130-140g respectively were selected.
The animals were maintained in the central animal house,
RMMC, Chidambaram 12:12 light:dark cycle at room
temperature 26±1°C. The animals were provided with
standard pellet diet “Gold Mohar” rat feed (Hindustan Lever
Company, Mumbai) and water ad libitum. The experiments
were conducted according to the ethical norms approved by
the Institutional Animal Ethics Committee (Proposal
No.744). The central animal house registration No:
160/1999/CPCSEA
The study was conducted under two experiments:
Experiment 1 – includes immature group <strong>of</strong> animals
Experiment 2 – includes mature group <strong>of</strong> animals
2.3. Experiment 1(Immature group)
Duration <strong>of</strong> drug administration: 15 days.
The animals were divided into three groups with six animals
in each.
Group I: Control rats received distilled water
intraperitoneally.
Group II: Rats treated with 10µg/kg <strong>of</strong> body weight <strong>of</strong>
Ghrelin administered intraperitoneally.
Group III: Rats treated with 20µg/kg <strong>of</strong> body weight <strong>of</strong>
Ghrelin administered intraperitoneally.
2.4. Experiment 2 (Mature group)
Duration <strong>of</strong> drug administration: 15 days.
The animals were divided into three groups with six animals
in each.
Group I: Control rats received distilled water
intraperitoneally.
Group II: Rats treated with 10µg/kg <strong>of</strong> body weight <strong>of</strong>
Ghrelin administered intraperitoneally.
Group III: Rats treated with 20µg/kg <strong>of</strong> body weight <strong>of</strong>
Ghrelin administered intraperitoneally.
2.5. Sample collection
After the experimental regimen, the animals were
sacrificed by cervical dislocation under mild ether anesthesia.
The body weights were recorded just before decapitation on
16 th day. The weight <strong>of</strong> the dissected ovaries was measured.
Ovaries from all animals were removed, cleared from
connective tissues and fat and then fixed in 10% buffered
formalin in labeled bottles.
2.6. Histological analysis
After the fixation <strong>of</strong> ovaries, they were directly
dehydrated in a graded series <strong>of</strong> ethanol, cleared in xylol and
embedded in paraffin wax. Thin sections (5μm), were cut
using a Rotatory microtome (LEICA RM2125RT) made in
China. Sections were stained with hematoxylin and eosin and
then viewed under a light microscope.
2.6.1. Histological procedures
After the extraction <strong>of</strong> the ovaries from the animal’s body,
they were weighed and then promptly treated with 10%

S. Antony Selvi et al, Int J Pharm Biomed Res 2012, 3(3), 141-146 143
formaldehyde (fixation) in order to preserve its structure and
molecular composition. After fixation, the piece <strong>of</strong> ovary was
dehydrated by bathing it successfully in graded mixture <strong>of</strong>
ethanol and water (70-100%). The ethanol was then replaced
with a solvent miscible with the embedding medium
(xylene). As the tissues were infiltrated with xylene, they
became transparent (clearing).
Once the tissue has been impregnated by xylene it was
placed in melted paraffin in an oven maintained at 58º-60°C
(embedding). The heat caused the solvent to evaporate and
the spaces within the tissues became filled with paraffin. The
tissue together with its impregnating paraffin hardened after
it had been taken out <strong>of</strong> the oven. The hard block containing
the tissue was then taken to the microtome and sectioned by
the microtome steel. The sections were then floated on water
and transferred to a glass slide and stained with heamatoxylin
and eosin. The slides were viewed under light microscope
with high power magnification. Light photograph <strong>of</strong>
histological slides <strong>of</strong> ovaries <strong>of</strong> both immature and mature
rats were taken by a Nikon camera attached to light
microscope.
(Table 1). In mature group, there is significant difference
with increase in body weight <strong>of</strong> low (10µg/kg) and optimum
dose (20µg/kg) received animals was observed when
compared to control group. The weight <strong>of</strong> ovaries in treated
animals show significant decrease when compared to control
was also noted. (Table 2).
Table 1
Body weight and weight <strong>of</strong> ovary in immature female group
S.No. Drug /dose Body wt (g) Wt <strong>of</strong> ovary (g)
1. Control 71.5 ± 3.6193 0.0366 ± 0.0051
2 Ghrelin (10µg/kg) 77.16 ± 2.136 * 0.025 ± 0.0054*
3 Ghrelin (20µ/kg) 85.33 ± 4.0368 * 0.0233 ± 0.0051*
Data are means ± SD. * P ˂ 0.05 compared with the control group
Table 2
Body weight and weight <strong>of</strong> ovary in mature female group
S.No. Drug/dose Body wt (g) Wt <strong>of</strong> ovary (g)
1. Control 119.16 ± 5.845 0.1100 ± 0.0109
2 Ghrelin (10µg/kg) 128.83 ± 5.269* 0.0750 ± 0.0104*
3 Ghrelin (20µ/kg) 140 ± 4.147* 0.0700 ±0.008*
Data are means ± SD. *P ˂ 0.05 compared with the control group
2.7. Statistical analysis
Body weight and weight <strong>of</strong> ovaries were expressed as
mean ± standard deviation (S.D) (n=6/group) .Differences
between groups were assessed by the analysis <strong>of</strong> variance
(ANOVA) using the SPSS version 17 s<strong>of</strong>tware package for
Windows. Statistical significance between groups was
determined by Tukey multiple comparison Post hoc test
values ˂ 0.05 compared with the appropriate control were
considered statistically significant.
3. RESULTS
Administration <strong>of</strong> <strong>ghrelin</strong> at the dose level 20µg/kg/ body
weight for 15 days resulted in structural disparity in ovarian
histology in comparison to that <strong>of</strong> the control and low dose
group.
The ovary showed follicular development <strong>of</strong> different
stages, but the number <strong>of</strong> mature graffian follicles and corpus
lutea were significantly reduced. Degeneration <strong>of</strong> corpus
luteum with occasional hemorrhage, increased atretic follicle
was also evident in <strong>ghrelin</strong> treated animals. Administration <strong>of</strong>
<strong>ghrelin</strong> at a dose level <strong>of</strong> 10μg/kg body weight /day revealed
similar but less pronounced structural abnormalities in
ovarian histology in comparison with that <strong>of</strong> optimum dose
treated animals. However immature group <strong>of</strong> animals did not
reveal any such structural ovarian changes in both the doses.
The treated animals in immature and mature group
showed decreased ovarian weight and increased bodyweight
in two dosages. In immature group <strong>of</strong> animals, compared to
control group there is significant differences with increase
body weight in low (10µg/kg) and optimum dose (20µg/kg)
received animals. There is a significant decrease in weight <strong>of</strong>
ovaries among treated when compared with control group.
4. DISCUSSION
The endocrine regulation <strong>of</strong> vertebrate reproduction is
achieved by the coordinated actions <strong>of</strong> multiple endocrine
factors mainly produced from the brain, pituitary, and
gonads. In addition to these, several other tissues including
the fat and gut produce factors that have reproductive effects.
Ghrelin is one such gut/brain <strong>hormone</strong> with species-specific
effects in the regulation <strong>of</strong> mammalian reproduction.
In the present study, body weight is increased in both the
experimental groups. In supportive with this result, it has
been recently shown that <strong>ghrelin</strong> induce a number <strong>of</strong>
biological responses at the central neuroendocrine level,
including stimulation <strong>of</strong> food intake and adiposity. It is
noteworthy that short- and long-term exogenous injection <strong>of</strong>
central and peripheral acyl <strong>ghrelin</strong> induced hyperphagia in
rats [26].
The weight <strong>of</strong> ovaries is significantly decreased in both
immature and mature group when compared to control. This
might be due to the suppression <strong>of</strong> follicular development
and ovarian sex <strong>hormone</strong>s secretion. It was reported that the
granulosa cells from <strong>ghrelin</strong>-treated rabbits secrete not only
less progesterone and estradiol but also less IGF-1 and
prostaglandin F than granulosa cells from untreated animals
[27]. Suppression <strong>of</strong> serum LH levels has been reported in
prepubertal male rats after central injection <strong>of</strong> <strong>ghrelin</strong> [17].
There is evidence in rats and humans that <strong>ghrelin</strong> can
suppress not only LH but also FSH secretion in males and
females [19].
In pertain with histoarchitecture, we emphasize that
<strong>ghrelin</strong> suppresses terminal stages <strong>of</strong> folliculogenesis
depending on varying doses in mature albino rats and the

S. Antony Selvi et al, Int J Pharm Biomed Res 2012, 3(3), 141-146 144
(A)
(B)
(C)
Fig.1. Light micrograph <strong>of</strong> ovarian sections <strong>of</strong> immature rats: control (A),
lower dosage (10µg/kg) (B), optimum dosage (20µg/kg) (C).
Ovary <strong>of</strong> control (A) shows ovarian tissue with primordial follicles and
primary follicles at various stages. No significant histological changes was
observed in both low and optimum dose treated animals
effects were more pronounced in the ovaries <strong>of</strong> optimum
dose treated animals <strong>of</strong> mature rats compared to those <strong>of</strong>
other groups. No significant changes were noted in ovaries <strong>of</strong>
immature rats (Fig.1).
The histological features <strong>of</strong> the ovary in optimum dose
treated rat exhibited structural changes (Fig.2) in comparison
to the control rat, suggesting some inhibitory actions <strong>of</strong>
<strong>ghrelin</strong> on the ovary. The ovary <strong>of</strong> the control showed all the
histological features <strong>of</strong> typical normal healthy rat. Each ovary
was enveloped in a germinal epithelium usually made up <strong>of</strong> a
single layer <strong>of</strong> cuboidal or low columnar cells. It contains a
cortex and a medulla. The cortex contains immature,
developing graffian follicles, atretic follicles and corpora
lutea.
In the present study, it was observed that the ovarian
histology shows structural disparity in graffian follicle, and
the number <strong>of</strong> mature graffian follicles and corpus lutea were
significantly reduced. As graffian follicle is indicative <strong>of</strong>
active folliculogenesis, disorganized cells indicates <strong>ghrelin</strong>
affects graffian follicular development which affects fertility.
An increase in the atretic follicles and decrease in corpora
lutea revealed the antiovulatory effect <strong>of</strong> <strong>ghrelin</strong>. Expression
<strong>of</strong> the functional <strong>ghrelin</strong> receptor has been reported in oocyte
as well as follicular, luteal, and surface epithelium and
interstitial hilus cells in rat ovary [11,28,29]. So we
hypothesis that intraperitoneal administration <strong>of</strong> <strong>ghrelin</strong>
might have action on the receptors present on follicular cells
and luteal cells and may have a negative influence on
fertility.
In supportive with this study, in vivo administration <strong>of</strong>
<strong>ghrelin</strong> in rats affects folliculogenesis as attested by
alterations <strong>of</strong> some morphometrical and intracellular indexes
in ovarian state. Indeed, it decreases the mean diameter <strong>of</strong>
follicles, the number <strong>of</strong> corpora lutea, luteal cells, and oocyte
and the diameter <strong>of</strong> the theca layer and the zona pellucida as
well as the whole ovarian volume in the treated animals [30].
Early destruction <strong>of</strong> corpus luteum with occasional
hemorrhage was observed in these animals. This in turn can
affect progesterone secretion .In cultured human granulosa
luteal cells <strong>ghrelin</strong> exert an inhibitory effect on
steroidogenesis (progesterone and estradiol production) in the
absence or in the presence <strong>of</strong> HCG by acting through its
functional GHS-R1a [31,32]. Ghrelin was not detected in
follicles regardless <strong>of</strong> their developmental stage; however
<strong>ghrelin</strong> expression was located in the ruptured follicle, the
corpus luteum [4].
Stromal tissues are prominent which implies reproductive
cells were replaced by it can be one <strong>of</strong> the factor which
affects fertility. There is congestion <strong>of</strong> blood vessels.
Ovulation can also get inhibited which may be due to direct
ovarian actions since administration <strong>of</strong> <strong>ghrelin</strong> inhibited
follicular development and altered the size distribution <strong>of</strong> the
follicles; observed follicles showed degeneration with intra
follicular hemorrhage. Ghrelin infusion decreased LH pulse
frequency, but not pulse amplitude in adult ovariectomized

S. Antony Selvi et al, Int J Pharm Biomed Res 2012, 3(3), 141-146 145
(A)
(B)
(C1)
(C2)
Fig.2. Light micrograph <strong>of</strong> ovarian sections <strong>of</strong> mature rats: control (A), lower dosage (10µg/kg) (B), optimum dosage (20µg/kg) (C) .Ovary <strong>of</strong> control (A)
shows ovarian tissue with primordial follicles and graffian follicles at various stages <strong>of</strong> maturation. Numerous corpus lutea and occasional corpus albicans are
seen (small arrow) While ovaries <strong>of</strong> lower dosage (B) shows mild degenerative changes like disorganized cells in graffian follicle. However, ovaries treated
with optimum optimum dosage (C1 & C2) show clearly that the terminal stages <strong>of</strong> folliculogenesis (Graffian follicle) and corpus luteum were affected, some <strong>of</strong>
which show haemorrhagium (big arrow). Occasional corpus luteum and congestion <strong>of</strong> blood vessels was also observed. Prominent Stromal tissues were also
noted (small arrow). Stained with Haematoxylin and eosin (viewed under high power magnification)
rhesus primates, suggesting that <strong>ghrelin</strong> could inhibit the
GnRH pulse activity [33].
With regard to the effect <strong>of</strong> systemic <strong>ghrelin</strong> on
gonadotropin secretion, several in vitro and in vivo animal
studies indicate that the <strong>ghrelin</strong> system negatively influences
the gonadal axis [4,34]. Acylated <strong>ghrelin</strong> suppresses
luteinizing <strong>hormone</strong> (LH) pulsatility in rodent, ovine, and
primate models [4,17,35-39].
Ghrelin treatment reduced the number <strong>of</strong> graffian follicle,
corpora lutea but increased the number <strong>of</strong> atretic follicles.
This resulted in a decrease in circulating progesterone
concentrations. Gonadotrophic <strong>hormone</strong>s (LH and FSH),
especially FSH accelerates the growth <strong>of</strong> immature ovarian
follicle; and the normal cyclicity depends on the ovarian
<strong>hormone</strong>, estrogen. As these <strong>hormone</strong>s are suppressed by the
actions <strong>of</strong> <strong>ghrelin</strong> there might be some alterations in
hormonal balance that led to ovarian dysfunctions resulted in
alterations in the histoarchitecture <strong>of</strong> the ovary that is
observed in the present study.
5. CONCLUSIONS
In overall view, the histology revealed the fact that
intraperitoneal administration <strong>of</strong> <strong>ghrelin</strong> on wistar strain
female albino rats affects folliculogenesis in different stages,
by acting on GHS-R present in ovarian cells especially on
follicular, luteal, surface epithelial cells and interstitial hilus
cells in rat ovary. It also causes reduction in the number and
degeneration <strong>of</strong> graffian follicles and corpus luteal cells that
can leads to infertility as it is the site <strong>of</strong> both egg and
<strong>hormone</strong> production. These effects were exhibited by the
<strong>ghrelin</strong> and were dose dependent.

S. Antony Selvi et al, Int J Pharm Biomed Res 2012, 3(3), 141-146 146
REFERENCES
[1] Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., Kangawa,
K., Nature 1999, 402, 656–660.
[2] Kojima, M., Kangawa, K., Physiol Rev 2005, 85, 495–522.
[3] Nishi, Y., Hiejima, H., Hosoda, H., Endocrinology 2005, 146, 2255–
2264.
[4] Barreiro, M.L., Tena-Sempere, M., Mol Cell Endocrino 2004, 226, 1– 9.
[5] Gualillo,O., Lago, F., Gomez-Reino, J., Casanueva, F.F., Dieguez, C.,
FEBS Letters 2003, 552 ,105–109.
[6] Vander Lely, A.J., Tschop, Heiman, M.L., Ghigo,E., Endocr Rev 2004,
25, 426–457.
[7] Sakata, I., Nakamura, K., Yamazaki, M., Matsubara, M., Hayashi, Y.,
Kangawa, K., et al., Peptides 2002, 23, 531–536.
[8] Lebrethon, M.C., Aganina, A., Fournier, M., Gerard, A., Parent, A.S.,
Bourguignon, J.P., J Neuroendocrinol 2007, 19, 181–188.
[9] Fernandez-Fernandez, R., Tena-Sempere, M., Navarro, V.M.,
Neuroendocrinology 2005, 82, 245 – 255.
[10] Nakazato,M., Masamitsu, Murakami, Noboru, Date, Yukari, Kojima,M.,
et al., Nature 2001, 409, 194-198.
[11] Caminos, J. E., Tena-Sempere, M., Gaytan, F., Endocrinology 2003,
144, 1594–1602,
[12] Zhang, W., Lei, Z., Su, J., Chen, S., Anim Reprod Sci 2008, 109, 356–
367.
[13] Miller, D.W., Harrison, J.L., Brown, Y. A., Reprod Biol Endocrinol
2005, 3, article 60.
[14] Gaytan, F., Barreiro, M.L., Chopin, L.K., J Clin Endocrinol Metab
2003, 88, 879–887.
[15] Sirotkin, A.V., Grossmann, R., Maria-Peon, M.T., Roa, J., Tena-
Sempere, M., Klein, S., Mol Cell Endocrinol 2006, 257-258, 15–25.
[16] Fernandez-Fernandez, R., Tena-Sempere, M., Roa, J Reproduction
2007, 133, 1223–1232.
[17] Fernandez-Fernández R, Tena-Sempere M, Aguilar E, Pinilla L.
Neurosci Lett 2004, 362, 103–107.
[18] Harrison, J.L., Miller, D.W., Findlay, P.A., Adam, C.L.,
Neuroendocrinology 2008, 87, 182–192.
[19] Vulliemoz, N.R., Xiao, E., Xia-Zhang, L., Rivier, J., Ferin, M.,
Endocrinology 2008, 149, 869–874.
[20] Lanfranco F., Bonelli, L., Baldi, M., Me, E., Broglio, F., Ghigo, E., Clin
Endocrinol Metab 2008, 93, 3633–3639.
[21] Forbes S.F., Li, X., Kinsey-Jones, J., O’Byrne, K., Neurosci Letters
2009, 460, 143–147.
[22] Lorenzi, T., Meli, R.D., Marzioni, M., Morroni, A., Baragli, M.,
Castellucci et al. Cytokine Growth Factor Rev 2009, 20, 137-152.
[23] Tena-Sempere, M., J Endocrinol Invest 2005, 28, 26–29.
[24] Garcia, M.C., Lopez, M., Alvarez, C.V., Casanueva, F., Tena-Sempere,
M., Dieguez, C., Reproduction 2007, 133, 531-540.
[25] Litwack,G., Tadhg Begley, Anthony Means, Bert O'Malley, Lynn
Riddiford , Armen, J., Tashjian., 2008, Oxford, UK, Elsevier Inc.
[26] Wren, A.M., Small, C.J., Ward, H.L., Murphy, K. G., Dakin, C. L.,
Taheri, S., Endocrinology 2000, 141, 4325-4328.
[27] Sirotkin, A.V., Rafay, J., Kotwica, J., Darlak, K., Valenzuela, F.,
Domest Anim Endocrinol 2009, 36, 162–172.
[28] Gaytan, F., Barreiro, M.L., Chopin, L.K., J Clin Endocrinol Metab,
2003, 88, 879–887.
[29] Gaytan,F., Morales,C., Barreiro, M.L., J Clin Endocrinol Metab 2005,
90, 1798–1804.
[30] Kheradmand, A., Roshangar, L., Taati, M., Sirotkin, A.V., Tissue Cell
2009, 41, 311–317.
[31] Viani, I., Vottero, A., Tassi, F., J Clin Endocrinol Metab 2008, 93,
1476–1481.
[32] Tropea, A., Tiberi, F., Minici, F., J Clin Endocrinol Metab 2007, 92,
3239–3245.
[33] Vulliemoz, N.R., Xiao, E., Xia-Zhang, L., Germond, M., Rivier, J.,
Ferin, M., J Clin Endocrinol Metab 2004, 89, 5718–5723.
[34] Tena-Sempere, M., Barreiro, M.L., Gonzalez, L.C, Gaytan, F., Zhang,
F.P., Caminos J.E. et al., Endocrinology 2002, 143, 717–725.
[35] Kawamura, K., Sato, N., Fukuda, J., Endocrinology 2003, 144, 2623–
2633.
[36] Furuta, M., Funabashi, T., Rimura, F., Biochem Biophys Res Commun
2001, 288, 780–785.
[37] Vulliemoz, N.R., Xiao, E., Xia-Zhang, L., Germond, M., Rivier ,J.,
Ferin, M., J Clin Endocrinol Metab 2004, 89, 5718–5723.
[38] Fernandez-Fernandez, R., Tena-Sempere, M., Navarro, V.M.,
Neuroendocrinology 2005, 82, 245–55.
[39] Iqbal, J., Kurose, Y., Canny, B., Clarke, I.J., Endocrinology 2006, 147,
510–519.