The Expression of a Mitochondria-Localized Glutamic Acid-Rich Protein (MGARP/OSAP) Is Under the Regulation of the HPG Axis

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The Expression of a Mitochondria-Localized Glutamic Acid-Rich Protein (MGARP/OSAP) Is Under the Regulation of the HPG Axis
NEUROENDOCRINOLOGY

                                                    The Expression of a Mitochondria-Localized Glutamic
                                                    Acid-Rich Protein (MGARP/OSAP) Is Under the
                                                    Regulation of the HPG Axis

                                                    Mingxue Zhou,* Yifeng Wang,* Shaoling Qi, Jian Wang, and Shuping Zhang
                                                    State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua
                                                    University, Beijing 100084, China

                                                    The hypothalamic-pituitary-gonadal (HPG) axis exerts a profound effect on animal development,
                                                    reproduction, and response to stress, and new insights into its complicated functional activities are
                                                    continuously being made. In the present study, by using immunohistochemical studies and dif-
                                                    ferent mouse models (ovariectomy and ob/ob mice), we systemically analyzed the expression of a
                                                    novel mitochondria-localized glutamic acid-rich protein (MGARP)/ovary-specific acid protein and
                                                    demonstrated that MGARP is under the regulation of the HPG axis. MGARP is highly enriched in
                                                    steroidogenic tissues and the visual system. Interestingly, its expression increases as mice develop.
                                                    Early in development, MGARP is mainly detected in the retina and adrenal gland. At this early
                                                    developmental stage, its expression is not detectable in the gonads, but its expression in the gonads
                                                    dramatically increases during the first 2– 4 wk after birth. Importantly, MGARP levels correlate with
                                                    estrogen levels in the ovaries during the estrous cycle, and estrogen regulates the expression of
                                                    MGARP in a tissue-specific manner and through a feedback regulatory mechanism. Functional
                                                    inhibition of GnRH with an antagonist strongly reduces MGARP levels, and knockout of leptin
                                                    (ob/ob) significantly reduces the MGARP expression in follicular granular cells. We proposed a
                                                    model that elucidates the role MGARP plays in the HPG axis. Within the HPG axis loop, MGARP
                                                    participates in hormone biosynthesis while being under the regulation of the hormones derived
                                                    from the HPG axis. (Endocrinology 152: 2311–2320, 2011)

    teroid hormones have a variety of metabolic activities,                                                           spermiogensis. Both LH and FSH participate in the regu-
S   including neuromodulatory, neuroendocrine, and neu-
roprotective effects, and they play crucial roles in mamma-
                                                                                                                      lation of the estrous cycle, menstrual cycle, gonad devel-
                                                                                                                      opment, and reproduction. Furthermore, androgen and
lian reproduction, development, aging, and stress responses                                                           estrogen use a feedback mechanism to regulate GnRH,
by acting on the brain and other organs (1, 2). All of these                                                          LH, and FSH biosynthesis (7). The HPG axis is an essential
activities of steroids are critically regulated by the hypo-                                                          and very complicated system throughout life, and there are
thalamic-pituitary-gonadal (HPG) axis (1, 3, 4). In the                                                               still many questions remaining to be answered.
HPG axis, GnRH, a factor secreted by the hypothalamus,                                                                    The mitochondria-localized glutamic acid-rich pro-
travels down the anterior portion of the pituitary via the                                                            tein (MGARP) is a novel mitochondrial protein identi-
hypophyseal portal system and binds to the receptors on                                                               fied by large-scale screenings for genes specifically ex-
the secretary cells of the adenohypophysis (5). Pulsatile                                                             pressed in the ovary, retina, cornea, and adrenal gland
release of GnRH stimulates the secretion of LH and FSH                                                                (8 –13). It was previously named as mouse ovary-spe-
in the adenohypophysis (6). LH regulates the synthesis of                                                             cific acidic protein and human corneal endothelium-
the gonadal hormones and ovulation, whereas FSH pro-                                                                  specific protein (8, 13), but its function was not sub-
motes ovarian follicle maturation, estrogen release, and                                                              stantially defined. In our previous report, we proposed

ISSN Print 0013-7227 ISSN Online 1945-7170                                                                            * M.Z. and Y.W. contributed equally to this work.
Printed in U.S.A.                                                                                                     Abbreviations: CNS, Central nervous system; E2, 17␤-Estradiol; E2-H, E2 high dose; E2-L,
Copyright © 2011 by The Endocrine Society                                                                             E2 low dose; GST, glutathione transferase; HE, hematoxylin and eosin; HPG, hypothalamic-
doi: 10.1210/en.2011-0050 Received January 18, 2011. Accepted March 8, 2011.                                          pituitary-gonadal; LGN, lateral geniculate nucleus; MGARP, mitochondria-localized glu-
First Published Online March 29, 2011                                                                                 tamic acid-rich protein; opt, optic tract; OVX, ovariectomy.

                                                                                                  Endocrinology, June 2011, 152(6):2311–2320                                         endo.endojournals.org            2311

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The Expression of a Mitochondria-Localized Glutamic Acid-Rich Protein (MGARP/OSAP) Is Under the Regulation of the HPG Axis
2312     Zhou et al.          MGARP/OSAP Mediated by Hormones in the HPG Axis                                                                             Endocrinology, June 2011, 152(6):2311–2320

an accordant and universal name, MGARP, for the pro-                                                             adjuvant and boostered weekly for 4 wk. The serum of the
tein in consideration of its enrichment with glutamic                                                            rabbit was collected 1 wk later, and the purity and specificity
acids and specific cellular localization (11). Our study                                                         of the antibody were analyzed by Western blotting and im-
                                                                                                                 munocytochemistry by using anti-GST, preimmunized serum
on MGARP in the retina showed robust levels of ex-
                                                                                                                 as control. The purified MGARP protein was used to do an-
pression in the inner segment of the photoreceptor,                                                              tigen preabsorption.
outer plexiform layer, and ganglion cell layer of the
retina (11). A reduction in MGARP expression results in                                                          Identification of the female mice estrous cycle
mitochondrial fragmentation and overexpression of                                                                   A vaginal cast-off cell smear, hematoxylin and eosin (HE)
MGARP with a deletion of the N terminus causes severe                                                            staining method was used to identify the estrous cycle of fe-
mitochondrial aggregation (10, 11). In Y-1 cells, knock-                                                         male mice. A dipped wet cotton bud with 0.9% isotonic so-
down of MGARP significantly inhibits 8-bromoad-                                                                  dium chloride was inserted into the vagina and rotated several
enosine-cAMP-induced progesterone production (10).                                                               times. Samples were smeared onto slices and fixed with meth-
                                                                                                                 anol. The vaginal cast-off cellular morphology was observed
The detailed regulatory mechanisms controlling MGARP
                                                                                                                 after HE staining.
expression, however, still remain to be elucidated. It is also
unclear how MGARP interacts with the different elements                                                          GnRH antagonist experiment
of the HPG axis, which includes the main components of                                                              ICR mice were divided into the following four groups: male
both the neurosecretory and steroidogenic systems.                                                               control group, male GnRH antagonist group, female control
   In this study, we systemically analyzed the expression                                                        group, and female GnRH antagonist group (n ⫽ 4 – 6/group).
and the regulatory network of MGARP by Western blot-                                                             Mice in the GnRH antagonist groups were treated with daily sc
ting and immunohistochemical analysis using different                                                            injections of 0.5 mg/kg cetrorelix acetate (14) for 7 d, whereas the
                                                                                                                 control groups were treated with the same volume of saline. The
mouse models and found that MGARP is predominantly
                                                                                                                 phase of the estrous cycle for all of the female mice was identified
expressed in the steroidogenic tissues and the compart-
                                                                                                                 by the vaginal cast-off cell smear test before and after cetrorelix
ments of the visual system. We also found that MGARP                                                             treatment.
expression increases as the gonads develop. It can be reg-
ulated by estrogen, GnRH, and leptin, all of which are                                                           Leptin intervention
regulators or effectors of the HPG axis. Our results suggest                                                         ICR mice were divided into the following six groups: male
that the interaction between MGARP and elements of the                                                           control group, male leptin low-dose group, male leptin high-dose
HPG axis forms a functional loop, with steroid hormones                                                          group, female control group, female leptin low-dose group, and
as the mediators.                                                                                                female leptin high-dose group (n ⫽ 3/group). A dose of either 100
                                                                                                                 or 500 ␮g/kg leptin was given daily for 5 d by ip injection. The
                                                                                                                 dose of 100 ␮g/kg leptin corresponds to the physiological dose
                                                                                                                 (15), and 500 ␮g/kg of leptin corresponds to a hyperphysiologi-
Materials and Methods                                                                                            cal dose. The control group was treated with the same volume of
                                                                                                                 saline. Euthanasia was performed on all mice (1% sodium pen-
Reagents and animals                                                                                             tobarbital). The phase of the estrous cycle for all the female mice
   17␤-Estradiol (E2) (E8875) and leptin (L3772) were pur-                                                       was identified by the vaginal cast-off cell smear test before and
chased from Sigma (St. Louis, MO). Cetrorelix acetate was from
                                                                                                                 after leptin treatment.
Shanghai Taishi Biotechnology Co., Ltd. (Shanghai, China). ICR
mice were bred by the Animal Facility of Tsinghua University.
Ob/ob mice were obtained from The Jackson Laboratory (Bar
                                                                                                                 Immunohistochemistry
Harbor, ME). All animal experiments were performed in com-                                                          Tissues and organs, except for the eyeballs, from each adult
pliance with the relevant laws and institutional guidelines. The                                                 mouse were fixed in 10% formalin for 24 h and embedded in
animal care procedures were institutionally reviewed a by the                                                    paraffin. Eyeballs were fixed in a special fixative solution (glacial
Institutional Animal Care and Use Committee of Tsinghua Uni-                                                     acetic acid:formalin:0.9% sodium chloride:75% alcohol, 1:2:7:
versity. The euthanasia was performed by adopting 1% sodium                                                      10). Two serial 5-␮m paraffin sections were used for immuno-
pentobarbital by ip injection.                                                                                   histochemical staining. Mouse MGARP antibody or GST anti-
                                                                                                                 body, used as a control, was added to the sections using a drop-
Antibody preparation                                                                                             wise technique.
   The full-length MGARP cDNA sequence was inserted into                                                            After the mice were euthanized, the tissues from each mouse
pGEX-4T-1 vector and transformed into the BL21 expression                                                        were removed, fixed for 24 h, and embedded in paraffin. Then
strain. Isopropyl ␤-D-1-thiogalactopyranoside (0.1 mM) was                                                       5-␮m sections were cut and stained using an immunohistochem-
used to induce the expression of a glutathione transferase (GST)-                                                ical method to observe and analyze MGARP expression. Addi-
MGARP fusion protein in Escherichia coli, and the protein was                                                    tionally, the same tissue types used for immunohistochemical
purified with an affinity column. A rabbit was inoculated with                                                   staining were also taken from the contralateral side of each
purified GST-MGARP fusion protein mixed with Freund’s                                                            mouse for Western blotting.

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The Expression of a Mitochondria-Localized Glutamic Acid-Rich Protein (MGARP/OSAP) Is Under the Regulation of the HPG Axis
Endocrinology, June 2011, 152(6):2311–2320                                                                                                                                          endo.endojournals.org            2313

                                                                                                                                                          Group, Inc., Chicago, IL), followed by a sec-
                                                                                                                                                          ondary antibody (goat antimouse IgG horse-
                                                                                                                                                          radish peroxidase-conjugated antibody,
                                                                                                                                                          1:5000; Zhongshan Golden Bridge). Protein
                                                                                                                                                          expression was detected with an enhanced
                                                                                                                                                          chemiluminescence detection system (Vigor-
                                                                                                                                                          ous, Beijing, China).

                                                                                                                                                          Surgical procedures and estrogen
                                                                                                                                                          treatment
                                                                                                                                                              Mice were weighed and anesthetized with
                                                                                                                                                          1% sodium pentobarbital by ip injection. Sur-
                                                                                                                                                          gical castration was performed through the
                                                                                                                                                          backside to gain bilateral access to the ovary.
                                                                                                                                                          After the ovaries were removed, the skin was
                                                                                                                                                          sutured with 3– 0 vicryl. Mice in the sham
                                                                                                                                                          group only had fatty tissue near the ovaries
                                                                                                                                                          removed as a control. Ten days after the op-
                                                                                                                                                          erations, mice were divided into the following
                                                                                                                                                          four groups: control group with ovariectomy
                                                                                                                                                          (OVX) (n ⫽ 5), OVX ⫹ E2 low-dose (E2-L)
                                                                                                                                                          group (n ⫽ 6, 10 ␮g/kg), OVX ⫹ E2 high-
                                                                                                                                                          dose (E2-H) group (n ⫽ 6, 100 ␮g/kg), and
                                                                                                                                                          sham treatment group (n ⫽ 5). Estrogen (E2)
                                                                                                                                                          was dissolved in 10% alcohol and 90% pea-
                                                                                                                                                          nut oil (16) and given daily by sc injections for
                                                                                                                                                          1 wk. The dose of 10 ␮g/kg of E2 corresponds
                                                                                                                                                          to the physiological dose, and 100 ␮g/kg of E2
FIG. 1. Immunohistochemical staining shows the expression pattern of MGARP in the visual
                                                                                                                                                          corresponds to a hyperphysiological dose.
tissues of ICR mice. A, Immunostaining of different regions in the eyeballs: retina (Re), optic nerve
(ON), cornea (Co), lens (Le), ciliary body (CB), and choroid and sclera (Ch/Sc). B, MGARP expression                                                      Mice in the control group and sham group
in the visual system of the brain: superficial gray layer of the superior collicullus (SuG), laterodorsal                                                 were sc injected with the same dose of vehicle
thalamic nucleus ventrolateral part (LDVL), LGN, optic chiasma (Ox), and opt. Opt ⫹ AP means                                                              (90% peanut oil and 10% alcohol).
that anti-MGARP antibody was previously preabsorbed with antigen. AP, Antigen preabsorption.
Images in white squares are magnification of areas enclosed by small white squares. Antibody                                                              Statistical analysis
labeling appears brown. Black and white scale bars, 50 and 200 ␮m, respectively.
                                                                                                                                           Data are presented as the means ⫾ SE
                                                                                                                                         and were analyzed by ANOVA, followed
Antigen preabsorption                                                                                                by Tukey’s test for multiple comparisons or Student’s t test.
   Anti-MGARP antiserum was diluted (1:10,000) with purified                                                         Differences are considered significant when P ⬍ 0.05.
MGARP-GST fusion protein dissolved in PBS with 1% BSA. The
protein level of purified MGARP-GST fusion protein is 5.2
mg/ml (determined by bicinchoninic acid assay; Pierce, Rockford,
IL). Anti-MGARP antiserum diluted (1:10,000) with PBS supple-
                                                                                                                     Results
mented with 1% BSA was set as positive control. Both of which
                                                                                                                     MGARP is highly enriched in the steroidogenic and
described above were incubated at 37 C for 1 h and followed by
standard immunohistochemistry procedure. The tissue sections                                                         visual systems
were also preincubated with 1% BSA in room temperature for 2 h.                                                          The systemic study on the MGARP expression in adult
                                                                                                                     mouse by immunohistochemistry demonstrated that, in
Western blotting                                                                                                     addition to the retina, MGARP is highly expressed in all
    Total protein was isolated from mice tissues. Tissues were                                                       other parts of the eyeball, including cornea, lens, ciliary
collected and homogenized in protein extraction buffer [50 mM                                                        body, sclera, and choroid. There was robust expression in
Tris-HCl (pH 7.4), 0.25 M NaCl, 1% Nonidet P-40, 1 mM EDTA,
                                                                                                                     the corneal epithelial cells, scleral fibroblast cells, epithe-
and 1% protease inhibitor cocktail]. The lysate was centrifuged
at 12,000 ⫻ g for 10 min, and the supernatant was collected. The                                                     lial cells in the lens, and pigment epithelial layer of the
supernatant (30 ␮g of protein) was resolved on a 10% SDS-                                                            ciliary body (Fig. 1A). The systemic expression profile also
PAGE gel and transferred onto nitrocellulose membranes. After                                                        showed that MGARP is highly expressed in the ovary,
being blocked with 5% nonfat milk, the membranes were probed                                                         testis, adrenal gland, eye, and brain, but not detectable in
with the primary mouse MGARP antibody (1:10,000) for 1 h,
                                                                                                                     heart, liver, spleen, lung, kidney, skeletal muscle, fat tis-
followed by a secondary antibody (goat antirabbit IgG horseradish
peroxidase-conjugated antibody, 1:5000; Zhongshan Golden                                                             sue, stomach, small intestine, uterus, pancreas, prostrate
Bridge, Beijing, China), or probed with the primary antibodies an-                                                   thymus, parathyroid gland, pituitary gland, and thyroid
tiactin (1:500; Sigma) and anti-GAPDH (1:4000; Proteintech                                                           gland by immunohistochemistry (Supplemental Fig. 1A,

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The Expression of a Mitochondria-Localized Glutamic Acid-Rich Protein (MGARP/OSAP) Is Under the Regulation of the HPG Axis
2314     Zhou et al.          MGARP/OSAP Mediated by Hormones in the HPG Axis                                                                             Endocrinology, June 2011, 152(6):2311–2320

published on The Endocrine Society’s Journals Online
web site at http://endo.endojournals.org). Using the anti-
gen-adsorbed MGARP antibody, the staining was com-
pletely eliminated (Supplemental Fig. 1B). Further analy-
sis indicated that MGARP was enriched in the lutein cells
of corpus luteum, theca cells, and granulosa cells of ovar-
ian follicles, Sertoli cells, and interstitial cells of mice testis
as well as the zona glomerulosa, zona fasciculata, and
zona reticularis of adrenal cortex. All these areas are made
up by steroidogenic cells (Supplemental Fig. 2). Most im-
portantly, our results showed that MGARP was detected
in different regions of the brain, including the optic chi-
asma, optic tract (opt), lateral geniculate nucleus (LGN),
laterodorsal thalamic nucleus ventrolateral part, and su-
perficial gray layer of the superior collicullus (SuG) (Fig.
1B). All of these comprise the main components of the
visual nervous system. Together, our observations indi-
cate that MGARP is enriched in steroidogenic tissues and
the visual system. Considering that the visual nervous sys-
tem, including the retina and related areas in the brain,
belongs to the central nervous system (CNS) as well as
responds to hormones, we hypothesized that MGARP is
potentially involved in the functional activities of the HPG
axis.

The expression of MGARP during mouse
development
    To understand the role of MGARP in development that is
critically regulated by the HPG axis, we studied MGARP
expression during steroidogenic tissue development. As
shown in Fig. 2, at postnatal d 1, MGARP is readily detected
in the adrenal gland and only slightly detected in the retina.                                                   FIG. 2. MGARP protein expression is associated with the progression
It was not detectable, however, in the mouse ovaries and                                                         of mouse development. Black and white scale bars, 50 and 200 ␮m,
                                                                                                                 respectively. A, Immunohistochemical staining of MGARP in the testes
testes at this stage. In the gonads, MGARP expression was                                                        (Te), ovaries (Ov), retinas (Re), and adrenal glands (AG) of mice at
clearly detected in pups 2– 4 wk after birth (Fig. 2, A–C).                                                      postnatal d 1 (P1), postnatal wk 2 (2W), 4W, and 8W. B, Western
In addition, we studied MGARP expression in more detail                                                          blotting of total proteins harvested from the Te, Ov, Re, and AG during
                                                                                                                 development. C, Column diagram indicates the quantitative analysis of
during the five phases of ovarian follicle development by
                                                                                                                 the Western blotting.
immunohistochemistry. The expression of MGARP in the
later phases was much higher than in the early phases
(Supplemental Fig. 3).                                                                                              To further determine the effects of sex hormones on
                                                                                                                 MGARP expression, we generated OVX mice to reduce
The effects of estrogen on MGARP expression                                                                      the endogenous estrogen or performed sc injection into the
   To study whether the expression of MGARP is regu-                                                             OVX mice with different doses of E2 to restore the estro-
lated by sex hormones and associated with steroidogenic                                                          gen level. The expression of MGARP was decreased in the
activity of the HPG axis, we examined MGARP expres-                                                              retina of the OVX mice compared with the sham group,
sion in female mice at different estrous cycle phases. We                                                        but the magnitude is not statistically significant, whereas
found that in ovaries, the expression of MGARP was sig-                                                          injection of E2 in OVX mice at a dose of 100 ␮g/kg could
nificantly higher during estrus and diestrus than during                                                         increase the expression of MGARP by 25% compared
proestrus and metestrus (Fig. 3, A–C). However, there was                                                        with the OVX group (Fig. 3D). In contrast, the expression
no significant fluctuation in MGARP expression in the                                                            of MGARP in the adrenal gland was increased in the OVX
mice adrenal glands and retinas at different estrous cycle                                                       mice compared with the sham group. Furthermore, injec-
phases (Fig. 3C).                                                                                                tion of E2 at a dose of 100 ␮g/kg could reduce the expres-

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The Expression of a Mitochondria-Localized Glutamic Acid-Rich Protein (MGARP/OSAP) Is Under the Regulation of the HPG Axis
Endocrinology, June 2011, 152(6):2311–2320                                                                                                                                         endo.endojournals.org            2315

                                                                                                                                                         in follicular cells of ovary was also
                                                                                                                                                         reduced, but it is not as significant as
                                                                                                                                                         that occurring in treated testes (Fig.
                                                                                                                                                         4A). As shown by Western blotting,
                                                                                                                                                         cetrorelix treatment led to the reduc-
                                                                                                                                                         tion in MGARP expression by 62% in
                                                                                                                                                         testes and 33% in ovaries compared
                                                                                                                                                         with the control groups (Fig. 4, A–C).
                                                                                                                                                         However, no difference was observed
                                                                                                                                                         in the retina and adrenal gland.

                                                                                                    The effects of leptin on MGARP
                                                                                                    expression
                                                                                                        To further examine a role of the HPG
                                                                                                    axis in regulating MGARP expression,
                                                                                                    we investigated the effects of leptin,
                                                                                                    which has been reported to stimulate the
                                                                                                    GnRH secretion through acting on the
                                                                                                    hypothalamus (17). As shown in Fig. 5, A
                                                                                                    and B, exogenous injection of leptin into
                                                                                                    female mice did not induce an obvious
                                                                                                    change in MGARP expression in the ova-
                                                                                                    ries. In the follicular granular cells of the
                                                                                                    sinusoid follicle, however, a low-dose
                                                                                                    of leptin (100 ␮g/kg) clearly increased
                                                                                                    MGARP expression compared with the
FIG. 3. MGARP expression fluctuates during the estrous cycle in the ovary, and its expression       control. To discount the effect of the es-
is estrogen independent in the retinas and adrenal glands. Black, white, and yellow scale bars,     trous cycle on MGARP expression, we
50, 200, and 500 ␮m, respectively. A, left panel, Proestrus (PE), estrus (OE), metestrus (ME),      carried out similar studies by using male
and diestrus (DE) in the estrous cycle of female mice are identified by HE staining of vaginal
cast-off cells. Right panel, Mice ovaries at different estrous cycle phases are immunostained
                                                                                                    mice. Injection of exogenous leptin at a
with MGARP antibody. B, Western blotting (WB) results of MGARP levels in mouse ovaries              dose of 100 ␮g/kg into adult male mice
(OV), retinas (Re), and adrenal glands (AG) at different estrous cycle phases. C, Quantitative      also increased MGARP expression by
analysis (column diagram) of MGARP levels in mouse ovaries (OV), retinas (Re), and adrenal
                                                                                                    45% in the testes compared with the con-
glands (AG) at different estrous cycle phases based on WB results (n ⫽ 5). D, left panel,
Retinas from sham, OVX, E2-L, and E2-H mouse groups are immunostained with anti-MGARP               trol, with a particularly higher stimula-
body. Middle and right panels, Western blotting results and quantitative analysis (column           tion in Leydig cells (Fig. 5, C and D).
diagram) of MGARP levels in the retinas from sham, OVX, E2-L, and E2-H mouse groups. E,                 Another model used in this study is the
left panel, Adrenal glands from sham, OVX, E2-L, and E2-H mouse groups are immunostained
with anti-MGARP body. Middle and right panels, Western blotting results and quantitative            female    leptin knockout (ob/ob) mice,
analysis (column diagram) of MGARP levels in the adrenal glands from sham, OVX, E2-L, and           which have been demonstrated to be in-
E2-H mouse groups.                                                                                  fertile (18). We studied the expression of
                                                                                                    MGARP in ovaries of ob/ob mice and
sion levels of MGARP by 21% in the adrenal gland com- wile-type C57 mice by immunohistochemistry. Consistently,
pared with the control of OVX group (Fig. 3E).                                  the overall expression of MGARP in ovaries of the ob/ob
                                                                                mice did not show significant differences, but its expression
The effects of functional inhibition of GnRH on                                 in follicular granular cells was markedly reduced compared
MGARP expression                                                                with the wild-type control mice (Fig. 5, E and F).
    Next, we studied MGARP expression by manipulation
of GnRH, a key factor in the HPG axis. Cetrorelix was
used in these tests, which is a GnRH antagonist that com- Discussion
petes the binding of GnRH to its receptors to inhibit go-
nadotropin level. The staining of MGARP in the cyto- We previously identified the MGARP gene by a microar-
plasm of Leydig cells and Sertoli cells of mice testes was ray from the mouse retina (11). Retina, a particularly ac-
reduced under the treatment of cetrorelix (GnRH antag- cessible part of the CNS, is critical for the capture of im-
onist) compared with the untreated control. Its expression ages and the response to light. These processes require the

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2316     Zhou et al.          MGARP/OSAP Mediated by Hormones in the HPG Axis                                                                             Endocrinology, June 2011, 152(6):2311–2320

                                                                                                   mone (estrogen, progesterone, and an-
                                                                                                   drogen) actions. Various physiological
                                                                                                   conditions, such as age, menstrual cy-
                                                                                                   cles, pregnancy, and menopause or an-
                                                                                                   dropause can affect vision (24). Studies
                                                                                                   have also shown the presence of sex ste-
                                                                                                   roid hormone receptors in various oc-
                                                                                                   ular tissues, such as the lens, retina,
                                                                                                   choroid, cornea, and ciliary body, and
                                                                                                   the response of the retina to sex steroid
                                                                                                   hormone action is similar to that of the
                                                                                                   CNS (24). With in situ hybridization
                                                                                                   techniques, estrogen receptor mRNA
                                                                                                   was detected in both the retina and
                                                                                                   brain (25, 26). The retina was also able
                                                                                                   to synthesize steroid hormones by the
                                                                                                   progesterone pathway (27). Consider-
                                                                                                   ing the CNS (brain and spinal cord) and
                                                                                                   retina are steroidogenic tissues (27–
                                                                                                   29), and steroid hormones are regu-
                                                                                                   lated by the HPG axis, our findings sug-
                                                                                                   gest a profound role of MGARP in the
FIG. 4. The GnRH antagonist cetrorelix acetate down-regulates MGARP levels in the testis
and ovary but not in the retinas and adrenal glands. Black and white scale bars, 50 and 200        regulatory loop of the HPG axis.
␮m, respectively. A, Immunostaining for MGARP in the mouse testes (Te), ovaries (Ov), retinas         However, why is MGARP predom-
(Re), and adrenal glands (AG) without [control (con)] and with GnRH antagonist [cetrorelix         inantly expressed within the visual sys-
acetate (Cetr), 0.5 mg/kg] treatment for 7 d. B, Total protein from comparable tissues
previously mentioned in A was harvested for Western blot analysis of MGARP. Each column
                                                                                                   tem? This is a critical question that is
represents one mouse. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. C, Column                   worth of further study. Here, we pro-
diagrams indicating the quantitative analysis of the Western blotting. *, P ⬍ 0.05; n ⫽ 4, 5,      pose several reasons. 1) The visual sys-
or 6.
                                                                                                   tem has higher demand for both energy
                                                                                                   and steroid hormones. 2) As part of the
collaboration of several different parts of the eyeball. To
                                                                             CNS, the visual system is the major regions of the animal
understand a potential role of MGARP in the visual sys-
                                                                             body to sense or receive the stimuli from the environment.
tem, we tested for MGARP expression in each part of the
                                                                             The visual perception is not simply a translation of stimuli
eyeball by immunohistochemistry. The results demon-
                                                                             and formation of the image on the retina and it should need
strated that, in addition to the retina, MGARP is also
                                                                             the eye and the related visual tissues in the brain to work
highly expressed in all other parts of the eyeball. To gather
                                                                             together in a very complicated way. This is a question
more information about MGARP, we conducted a sys-
                                                                             scientists in this field have long struggled to explore. Our
temic analysis of MGARP expression by immunohisto-
chemical staining. The results showed that MGARP is observation that MGARP is highly expressed in visual
highly expressed in steroidogenic and nervous tissues, es- system implies that MGARP may play critical function
pecially in areas that make up the main components of the in transmitting the stimuli through responding to the
visual nervous system. The optic chiasma, opt, and LGN steroid hormones or through regulating hormone syn-
are the main components of the visual pathway in brain. thesis. 3) The expression difference in the brain reflects
These regions are responsible for transmitting visual in- that MGARP expression is under the regulation of dif-
formation from the eyeball to the optic center in the brain. ferent hormones in a direct or indirect manner, because
The superficial gray layer of the superior collicullus is re- different regions of the brain produce distinct hormones
sponsible for providing a powerful excitatory input to the and those hormones always function in different tissues
intermediate layer, which plays a critical role in initiating through circulation. It can be considered that HPG axis
rapid orienting movements of the eye (19 –23). Thus, most may steer the entire process through regulating the ex-
of the regions positive for MGARP expression are directly pression level of MGARP. Certainly, all these need more
or indirectly involved in visual processing. The visual sys- studies to be clearly addressed.
tem is a special system that has long been used as a model                      The HPG axis plays an active and essential role in mam-
for CNS developmental and functional studies of sex hor- malian development via modulating various hormones.

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Endocrinology, June 2011, 152(6):2311–2320                                                                                                                                         endo.endojournals.org            2317

                                                                                                    and MGARP is less expressed in the
                                                                                                    early phase of follicular development
                                                                                                    than in other phases (31). Our results
                                                                                                    of the MGARP expression during the
                                                                                                    five phases of ovarian follicle develop-
                                                                                                    ment indicate that MGARP expression
                                                                                                    is gradually increased during follicle de-
                                                                                                    velopment, which is most likely regu-
                                                                                                    lated by gonadotropin.
                                                                                                        One physiological function of the
                                                                                                    HPG axis is to regulate the synthesis of
                                                                                                    sex hormones. Conversely, the sex hor-
                                                                                                    mones regulate the HPG axis through a
                                                                                                    feedback regulatory mechanism. Estro-
                                                                                                    gen is a sex hormone that not only af-
                                                                                                    fects the sex organs, but it also affects
                                                                                                    the structure and function of the ner-
                                                                                                    vous system, because its receptors are
                                                                                                    expressed in the brain regions that are
                                                                                                    involved in sex differentiation and mat-
                                                                                                    uration (1). To determine whether the
                                                                                                    expression of MGARP is involved in
                                                                                                    the steroidogenic activity of the HPG
FIG. 5. MGARP expression is stimulated by leptin (Lep) and reduced in the granulosa cells of
ob/ob mice. Black and white scale bars, 50 and 200 ␮m, respectively. A, Western blot analysis
                                                                                                    axis, we used a female mouse model,
of MGARP levels in mouse ovaries (Ov) without or with leptin treatment for 5 d. Column              because they have a clear estrous cycle
diagram shows the quantitative analysis based on the Western blotting. *, P ⬍ 0.05;                 that is linked to the endogenous fluctu-
n ⫽ 3. Leptin-L, Leptin low dose (100 ␮g/kg); Leptin-H, leptin high dose (500 ␮g/kg). B,
                                                                                                    ations of sex hormones. In estrous cy-
Immunostaining shows that leptin treatment increases MGARP expression in the follicular
granular cells of the antral follicle from ICR mice compared with controls. Black arrowhead         cles, estrogen and progestin levels are
indicates follicular granular cells. C, Western blot analysis of MGARP levels in mouse testes       dominant during estrous and diestrous
(Te) without or with leptin treatment for 5 d. Column diagram shows the quantitative analysis       phase, thus, the MGARP expression
based on the Western blotting. *, P ⬍ 0.05; n ⫽ 3. D, Immunostaining shows that leptin
treatment increases MGARP expression in the Te of ICR mice, specifically in the Leydig cells        change in ovaries at estrous cycle suggests
compared with controls. Black arrowhead indicates Leydig cells. E, Western blot analysis of         that MGARP expression is correlated
MGARP expression in the Ov from normal C57 mice [control (con)] and ob/ob (leptin                   with the levels of estrogen and progesto-
knockout) mice. Column diagram shows the quantitative analysis based on the Western
blotting result (n ⫽ 3). F, Immunostaining shows that MGARP expression is reduced in the
                                                                                                    gen in the ovary. It is reasonable that no
granulosa cells (1, 3) and interstitial cells (2, 4) of ob/ob mice ovaries compared with controls.  significant change in MGARP expression
Black arrowheads indicate granulosa cells. C, Control; L, leptin low dose; H, leptin high dose.     can be found in the adrenal gland and
                                                                                                    retina during the estrous cycle, because
Observations of MGARP expression in mice retinas and
                                                                                the estrous cycle mainly affects the fluctuation of sex hor-
adrenal glands during mouse development indicate that
                                                                                mones in the gonads. Together, our findings indicate that
MGARP plays an early role in the development of the
                                                                                the expression of MGARP is regulated by steroid hor-
organs. In the gonads, MGARP expression was clearly
                                                                                mones in a tissue-specific manner.
detected in pups only 2– 4 wk after birth. We speculate that
                                                                                   OVX mice provide a model to monitor the effects of
as the levels of sex hormones increase in the gonads, they
stimulate the expression of MGARP. The increased ex-                            estrogen   under a reduced background. Our results indi-
pression of MGARP, in turn, directs the biosynthesis of cate that estrogen in OVX mice exerts opposite effects on
distinct sex hormones in the gonads and facilitates the MGARP expression in the retina and the adrenal gland,
development of the sex organs.                                                  further suggesting a tissue-specific regulatory mechanism
    During the development of mice ovaries, many ovar- that controls MGARP expression. The decreased expres-
ian follicles are involved in the process of maturation, sion of MGARP in the retina is likely due to a direct effect
whereas only a few of them named dominant follicles of estrogen reduction after removal of the ovaries. Con-
will eventually develop into mature ova (30). It has been versely, the increased expression of MGARP in the adrenal
reported that MGARP mRNA is up-regulated in dom- gland may be due to the negative feedback mechanism in
inant follicles compared with the subordinate follicles, the HPG axis that regulates estrogen levels (32). Simi-

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2318     Zhou et al.          MGARP/OSAP Mediated by Hormones in the HPG Axis                                                                             Endocrinology, June 2011, 152(6):2311–2320

                                                                                       sion by stimulating the secretion of GnRH via
                                                                                       the HPG axis.
                                                                                          Female leptin knockout (ob/ob) mice have
                                                                                       been demonstrated to be infertile, and leptin
                                                                                       treatment can restore the reproductive ability
                                                                                       of the ob/ob mice through reactivating the
                                                                                       HPG axis (36 –38). Consistently, our results
                                                                                       demonstrated that the overall expression of
                                                                                       MGARP in ovaries of the ob/ob mice did not
                                                                                       show significant difference, but its expression
                                                                                       in follicular granular cells was markedly re-
                                                                                       duced. Considering that estrogen is a major
FIG. 6. Model demonstrating the potential role of MGARP in the HPG axis. Solid         steroid hormone secreted by follicular granular
arrows represent known pathways, whereas dashed arrows represent proposed              cells, these results strongly support the belief
pathways. StAR, Steroidogenic acute regulatory protein; PKA, protein kinase A.
                                                                                       that MGARP is under the regulation of specific
                                                                                       hormones in the HPG axis. Based on these find-
larly, the sc injection of endogenous estrogen into OVX
                                                                          ings, we would suggest a mechanism by which leptin reg-
mouse also exerts the opposite effect on the expression
                                                                          ulates various reproductive events. We speculate that lep-
of MGARP in the retina and the adrenal gland. In mice,
                                                                          tin stimulates MGARP expression and steroidogenesis in
the adrenal gland may function as one of the major
                                                                          follicular granular cells, followed by the restoration of
organs that secrete sex hormones by increasing the ex-
                                                                          estrogen levels, the ability of ob/ob mice to reproduce and
pression of MGARP in the adrenal cortex once the ova-
                                                                          the ability of GnRH-deficient mice to ovulate (36).
ries have been removed.
                                                                               Mitochondria are the initial site of steroid biosynthesis.
    The hypothalamus is the center that controls the secre-
                                                                          Steroidogenic acute regulatory protein promotes the trans-
tion of steroid hormones by releasing a major steroid stim-
                                                                          portation of cholesterol from the mitochondrial inner mem-
ulator, GnRH, which is also a major regulator in the HPG
                                                                          brane to the outer membrane and accelerates the synthesis of
axis (6). GnRH binds to receptors on secretary cells of the
                                                                          steroid hormones (39, 40). Steroidogenic acute regulatory
adenohypophysis (5). Pulsatile release of GnRH can stim-
                                                                          protein is regulated by cAMP, a major second messenger of
ulate the secretion of LH and FSH in the adenohypophysis
(6). LH regulates estrogen synthesis, whereas FSH pro- the gonadotropins (41). MGARP is a mitochondrial trans-
motes estrogen release. Cetrorelix, a GnRH antagonist, membrane protein that functions during steroidogenesis
can inhibit the level of gonadotropin by competing with (10). The properties of its localization and unfolded struc-
GnRH for the binding sites on its receptors (14, 33, 34). ture suggest that MGARP may play a critical role in trans-
From our results, we can clearly see that disruption of porting the precursors for synthesizing the steroid hor-
GnRH activity by cetrorelix decreases MGARP expres- mones into the mitochondria or helping the synthesized
sion. These results imply that functional inhibition of steroid hormones export out of the mitochondria. Given
GnRH by cetrorelix may interfere with the regulatory loop all of these points, we proposed a model that summarizes
in the HPG axis and lead to the suppression of gonado- and elucidates the role MGARP plays in the HPG axis (Fig.
tropin, LH, and FSH production. These events would then 6). Within the HPG axis loop, MGARP participates in
cause the down-regulation of MGARP expression. How- hormone biosynthesis while being under the regulation of
ever, no difference was observed in the retina and adrenal the hormones derived from the HPG axis.
gland, suggesting that MGARP expression in these tissues                       In summary, our study revealed a regulatory loop
is not affected as strongly and quickly as that in gonads, or mechanism that exists between MGARP expression and
MGARP expression is regulated by tissue-specific factors. steroidogenesis in the HPG axis. Additionally, we dem-
    Leptin can stimulate GnRH secretion through acting on onstrated that MGARP is highly expressed both in the
the hypothalamus (35). Our results showed that exoge- visual nervous system and steroidogenesic tissues.
nous injection of leptin into female mice clearly increased MGARP is regulated in a development-dependent and tis-
the MGARP expression in follicular granular cells of an- sue-specific manner. This also implies that an animal’s
tral follicle, especially under the treatment of lower dose of body keeps the balance of hormone levels by combinatory
leptin (100 ␮g/kg). Similarly, exogenous injection of leptin mechanisms that are both feedback regulatory and tissue
into male mice could also stimulate MGARP expression specific. The coordination between steroid hormones and
by 45% in the testis, much higher than in ovary. These neuronal control regulates important events within the
results suggest that leptin can induce the MGARP expres- animal body, including organogenesis, growth, aging, the

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Endocrinology, June 2011, 152(6):2311–2320                                                                                                                                         endo.endojournals.org            2319

stress response, and the clearing of most diseases. Thus,                                                                  Teranishi T, Hattori Y, Furuya M, Higuchi T, Asai S, Kim SH,
                                                                                                                           Miyakoshi K, Yoshimura Y 2009 Expression of ovary-specific
this finding demonstrating the involvement of MGARP in
                                                                                                                           acidic protein in steroidogenic tissues: a possible role in steroido-
the HPG axis will be of great biological and clinical                                                                      genesis. Endocrinology 150:3353–3359
significance.                                                                                                       11.    Qi S, Wang Y, Zhou M, Ge Y, Yan Y, Wang J, Zhang SS, Zhang S 2010
                                                                                                                           A mitochondria-localized glutamic acid-rich protein (MGARP/OSAP)
                                                                                                                           is highly expressed in retina that exhibits a large area of intrinsic dis-
                                                                                                                           order. Mol Biol Rep 10.1007/s11033-010-9948-x
Acknowledgments                                                                                                     12.    Bailey MJ, Coon SL, Carter DA, Humphries A, Kim JS, Shi Q,
                                                                                                                           Gaildrat P, Morin F, Ganguly S, Hogenesch JB, Weller JL, Rath MF,
We thank Prof. Bruce S. McEwen (Laboratory of Neuroendo-                                                                   Møller M, Baler R, Sugden D, Rangel ZG, Munson PJ, Klein DC
crinology, Rockefeller University, New York, NY), Prof. Hon-                                                               2009 Night/day changes in pineal expression of ⬎600 genes: central
                                                                                                                           role of adrenergic/cAMP signaling. J Biol Chem 284:7606 –7622
gjun Song (Departments of Neurology and Neuroscience, Johns                                                         13.    Kinouchi R, Kinouchi T, Hamamoto T, Saito T, Tavares A, Tsuru
Hopkins University School of Medicine, Baltimore, MD), and                                                                 T, Yamagami S 2006 Distribution of CESP-1 protein in the corneal
Dr. Shaoyong Chen (Beth Israel Deaconess Medical Center, Har-                                                              endothelium and other tissues. Invest Ophthalmol Vis Sci 47:1397–
vard Medical School, Boston, MA) for constructive suggestions                                                              1403
and reading our manuscript. We also thank Prof. Peng Li (School                                                     14.    Meirow D, Assad G, Dor J, Rabinovici J 2004 The GnRH antagonist
                                                                                                                           cetrorelix reduces cyclophosphamide-induced ovarian follicular de-
of Life Sciences, Tsinghua University) for the help with the ob/ob                                                         struction in mice. Hum Reprod 19:1294 –1299
mice.                                                                                                               15.    Watanobe H, Suda T, Wikberg JE, Schiöth HB 1999 Evidence that
                                                                                                                           physiological levels of circulating leptin exert a stimulatory effect on
   Address all correspondence and requests for reprints to:                                                                luteinizing hormone and prolactin surges in rats. Biochem Biophys
Shuping Zhang, School of Life Sciences, Tsinghua University,                                                               Res Commun 263:162–165
Beijing 100084, China. E-mail: bczhang@tsinghua.edu.cn.                                                             16.    Fujita T, Kawata T, Tokimasa C, Tanne K 2001 Influence of oes-
                                                                                                                           trogen and androgen on modelling of the mandibular condylar bone
   This work was supported by funds from the State Key Lab-
                                                                                                                           in ovariectomized and orchiectomized growing mice. Arch Oral Biol
oratory of Biomembrane and Membrane Biotechnology (No.                                                                     46:57– 65
19881880001), the National Basic Research Program (also                                                             17.    Nagatani S, Guthikonda P, Thompson RC, Tsukamura H, Maeda
called the 973 Program) of China Grants 2006CB705700,                                                                      KI, Foster DL 1998 Evidence for GnRH regulation by leptin: leptin
and National Natural Science Foundation of China Grants                                                                    administration prevents reduced pulsatile LH secretion during fast-
                                                                                                                           ing. Neuroendocrinology 67:370 –376
30671036 and 30971513.                                                                                              18.    Chehab FF, Lim ME, Lu R 1996 Correction of the sterility defect in
   Disclosure Summary: The authors have nothing to disclose.                                                               homozygous obese female mice by treatment with the human re-
                                                                                                                           combinant leptin. Nat Genet 12:318 –320
                                                                                                                    19.    May PJ 2006 The mammalian superior colliculus: laminar structure
                                                                                                                           and connections. Prog Brain Res 151:321–378
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