Human Reproduction vol 11 no.9 pp.1952-1957, 1996
An aminopeptidase inhibitor, bestatin, enhances
gonadotrophin-stimulated ovulation in mice
Kimihiko Nakamura 1 , Hiroshi Fujiwara1>s,
Takahiro Nakayama 1 , Yasuo Goto1,
Toshiyuki Tachibana1, Hiroshi Suginami4,
Masamichi Ueda 2 , Michiyuki Maeda3 and
Takahide Mori 1
'Department of Gynecology and Obstetrics, Faculty of Medicine,
Institute for Virus Research and 3Chest Disease Research Institute,
Kyoto University, Sakyo-ku and 4Kyoto National Hospital,
Fushimi-ku, Kyoto, Japan
2
^ o whom correspondence should be addressed
The role of aminopeptidases in follicular growth and/or
ovulation in vivo was examined. We injected an inhibitor
of cell-surface aminopeptidases, bestatin (4 mg/ml, 100 fil),
i.p. four times during 2 days into 20 day old female
ICR mice, hi which follicular growth and ovulation were
stimulated by pregnant mare's serum gonadotrophin
(PMSG, 5 IU) and human chorionic gonadotrophin
(HCG, 5 IU). The number of ovulated oocytes was
estimated by counting the number of oocytes in the
oviduct 19 h after HCG injection. The mean ± SD number
of ovulated oocytes in bestatin-treated mice was significantly higher than that hi control mice [47.00 ± 18.13
(n = 26) versus 35.90 ± 10.14 (n = 28), P < 0.01]. To
confirm the direct effect of bestatin on the ovary, bestatin
(2 mg/ml, 3 |Xl> or its stereoisomer (2 mg/ml, 3 jil) with
very weak inhibitory activity was unilaterally injected into
the ovarian bursa 24 h before the administration of PMSG.
As a control, buffer (3 \il) was injected into the contralateral
bursa. In some experiments, bestatin (2 mg/ml, 3 (ll) was
injected just before HCG administration. The administration of bestatin via the ovarian bursa prior to PMSG
administration significantly increased the number of
ovulated oocytes per oviduct from the treated compared
with the contralateral ovary [23.70 ± 9.61 versus 17.10 ±
5.83 (n = 25), P < 0.01], whereas its stereoisomer elicited
no significant effects. The administration of bestatin just
before HCG administration also had no effect These
findings indicate that membrane-bound peptidase(s) present on murine ovarian cells is an important regulating
factor(s) of follicular growth and/or ovulation.
Key words: aminopeptidase/bestatin/folhculogenesis
Introduction
We reported previously that human theca intema cells express
aminopeptidase N (AP-N; EC 3.4.11.2) during folliculogenesis
(Fujiwara et al, 1992a, 1993). We also showed that AP-N is
1952
expressed on small luteal cells after ovulation, indicating that
AP-N is a differentiation marker of human theca intema and
small luteal cells (Fujiwara et al, 1992a). We then found that
another membrane-bound peptidase, dipeptidyl peptidase IV
(EC 3.4.14), is expressed on human luteal cells, and showed
that it is a luteinization marker of both granulosa and thecal
cells (Fujiwara et al., 1992b). Because AP-N and dipeptidyl
peptidase IV are membrane-bound peptidases and their catalytic
sites are extracellular, they can regulate the extracellular
peptide concentrations in the follicles and corpora lutea. It is
generally accepted that several peptides, including growth
factors and cytokines, are involved in folliculogenesis in
an autocrine and/or paracrine fashion (Hsueh et al, 1989).
Therefore, we proposed that these membrane-bound peptidases
play a role in folliculogenesis and luteal function by regulating
the extracellular peptide concentrations (Fujiwara et al.,
1992b).
Kenny et al. (1989) have speculated that cell-surface
peptidases play a key role in the control of growth and
differentiation of many cellular systems by modulating the
activity of peptide factors and regulating their access to
adjacent cells, although an adequate cellular system model(s)
has not yet been presented. Several membrane-bound peptidases have been identified as haematopoietic cell markers.
They are also thought to be involved in haematopoietic cell
differentiation. However, the precise physiological roles of the
membrane-bound peptidases have not been clarified (Shipp
and Look, 1993). In the ovary, the granulosa and thecal cells
show dynamic changes, including cellular growth, differentiation and regression, during the menstrual cycle. Thus, this
reproductive organ can provide a good model with which to
investigate the notion that membrane-bound peptidases are
involved in cellular growth and differentiation.
In this study, we found that the murine ovary also expressed
a membrane-bound aminopeptidase. Therefore, we investigated
the physiological role of membrane-bound peptidase(s) in
folliculogenesis in vivo by giving mice i.p. or ovarian bursal
injections of a peptidase inhibitor.
Materials and methods
Reagents
Pregnant mare's serum gonadotrophin (PMSG) and human chononic
gonadotrophin (HCG) were purchased from Teikoku Zoki Co. (Tokyo,
Japan), t h e rat monoclonal antibody, ER-BMDM1, which recognizes
a 160 leDa cell membrane aminopeptidase corresponding to human
AP-N/CD 13 antigen, was obtained from Serotec Ltd (Oxford, UK)
(Leenen et al, 1992). The rat monoclonal antibody, RHIg-87, which
recognizes hamster immunoglobulin (Ig) G, was obtained from Cosmo
© European Society for Human Reproduction and Embryology
Bestatin enhances ovulatlon in mice
Bio Co. Ltd (Tokyo, Japan) and was used for negative control staining
A fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG
polyclonal antibody pre-absorbed with mouse Ig was purchased from
Caltag Lab. Inc. (South San Francisco, CA, USA). L-Leucyl-p"naphthylamide hydrochlonde was obtained from Nacalai Tesque Inc.
(Kyoto, Japan), and Fast Blue Salt BN from Sigma (St Louis, MO,
USA). Bestatin {[(2S, 3R)-3-amino-2-hydroxy-4-phenylbutanoyl](S)-leucme}, an inhibitor of AP-N (Rich et al, 1984), and its
stereoisomer {[(2R, 3S)-3-amino-2-hydroxy-4-phenylbutanoyl]-(S)leucine}, which very weakly inhibits peptidase acnvity (Suda et al,
1976), were gifts from Nihon Kayaku Co. Ltd (Tokyo, Japan). Animals
were anaesthetized with nembutal (Dainabot Co., Osaka, Japan)
Indirect immunqfluorescence staining of ovarian tissues
Ovarian tissues were indirectly stained by lmmunofluorescence, as
described previously (Fujiwara et aL, 1994). Immature female ICR
mice were purchased from Charles River Japan Inc. (Kanagawa,
Japan). Fresh ovaries were obtained from immature ICR mice aged
6 (n = 10), 17 (n = 9), 28 (n = 10) and 35 days (n = 8).
The ovaries were embedded m OCT compound (Tissue-Tec, Miles
Scientific, Naperville, IL, USA), snap-frozen in liquid nitrogen and
stored at —80°C. Frozen ovarian tissues were sliced into 8 (im thick
sections using a cryostat microtome Cryocut 1800 (Reichert-Jung,
Heidelberg, Germany), then immediately air-dned and fixed in acetone
at -20°C for 5 min ER-BMDM1 antibody was diluted with RPMI1640 medium (Flow Labs, Irvine, UK) containing 10% fetal calf
serum (FCS; Flow Labs, Mclean, VA, USA) and 0 1% NaN3 to 1 100
(2 |ig/ml). For the negative control, RHlg-87 antibody was also
diluted with the same culture medium containing 10% FCS and 0.1%
NaN3 to 1:50 (2 H-g/ml). These first antibodies were added to ovarian
sections and incubated for 60 mm in a moist chamber at room
temperature. After washing the sbdes with phosphate-buffered saline
(PBS) three times, developing antibody diluted 140 with the same
culture medium was added and incubated for 30 mm at room
temperature in the dark. The slides were washed three times with
PBS and mounted with an agent (Perma Fluor Aqueous Mounting
Medium, Immunon, Pittsburgh, PA, USA) that reduces fluorescence
fading The slides were examined under a fluorescence microscope
(Nikon, Tokyo, Japan) by two individuals within 6 h, as described
by Fujiwara et aL (1994). The intensity of the antigen expression
was graded in three scores based on the fluorescence intensity (- =
none; + = weak; + + = intense). An ovary obtained from a 17 day
old mouse, the fluorescence intensity of which was weak, was used
as the standard visual assessment of fluorescence intensity throughout
this study. If judgements differed, the lower value was selected
Histochemical staining
Fresh ovaries from 6 (n = 10), 17 (n = 9), 28 (n = 11) and 35 (n =
9) day old mice were obtained Frozen sections for histochemical
staining were processed as described above. Peptidase activity was
examined by removing leucine from L-leucyl-p"-naphthylamide hydrochlonde as described by Fujiwara et al. (1992a), with some minor
modifications. Briefly, 5 mg L-leucyl-pVnaphthylamide hydrochlonde
were dissolved in 100 |Xl AW-dimethylformamide Fast Blue Salt BN
(5 mg) was dissolved in 10 ml 0.1 M PBS, pH 7 3. The two solutions
were mixed and passed through a 0.22 |im membrane filter just before
staining The filtrate was added to each section, and after an incubation
for 40 min at 37°C, the reaction was stopped by extensive washes
with PBS. Slides were mounted in glycerol-PBS (1:1, v/v) and
observed under a light microscope As negative controls, cryostat
sections were incubated with substrate-free medium (Hartel et al,
1988) The staining intensity of the peptidase activity was graded in
three scores (- = none; + = weak; + + = intense) and assessed by
two individuals. The standard visual assessment of staining intensity
throughout this study was performed as described above
I.p. and ovarian bursal injection of bestatin and the induction of
follicular growth and ovulation by PMSG and HCG
Some 17 day old immature female ICR mice were purchased from
Charles River Japan Inc., housed under controlled lighting (14 h of
light, 10 h of darkness), and given water and food ad libitum.
At 19:00 h on day 20, 07:00 and 19 00 h on day 21 and 0700 h
on day 22 bestatin [4 mg/ml dissolved with PBS containing 0 1%
bovine serum albumin (BSA), 100 |il] was injected I p into immature
mice. As a control, PBS containing 0.1% BSA (100 u.1) was
administered i.p. At 17:00 h on day 21, the development of multiple
follicles was induced by an i p injection of PMSG (5 IU), and at
17 00 h on day 23 HCG (5 IU) was injected i p. to induce ovulation,
as descnbed by Goto et aL (1992).
For the ovanan bursal mjection, the mice were anaesthetized with
an i.p. mjection of nembutal (0.25 mg/body) at 1700 h on day 20
Using a dissecting microscope (Olympus, Tokyo, Japan), one of
the ovanes was externalized via a lumbosacral incision Bestatin
(2 mg/ml) or its stereoisomer (2 mg/ml) was injected (3 |xl) into the
ovanan bursa, the side of which was chosen at random, using a fine
Pasteur pipette. The local mjection was confirmed by swelling of
the bursa PBS (3 (il) containing 0 1% BSA was injected into the
contralatera] bursa as a control Thereafter, the ovaries were replaced
in the abdominal cavity and the skin was sutured. At 17:00 h on
the following day (day 21), the developing follicles were induced by
an i.p injection of PMSG (5 IU), and at 1700 h on day 23, HCG
(5 IU) was injected I p. to induce ovulation as descnbed. In some
animals an lntrabursal mjection of bestatin (2 mg/ml) was given just
before HCG administration.
Counting the ovulated oocytes
At 12.00 h on day 24 the mice were killed by cervical dislocation
The number of ovulated oocytes per ovary was scored as the number
of oocytes in the oviduct by scratching, as described previously
(Natsuyama et al, 1993).
Statistical analysis
The difference in the fluorescence and the staining intensity scores
of aminopeptidase activity among groups were analysed by the
Kruskal-Wallis test followed by the Mann-Whitney test The numbers
of ovulated oocytes are presented as means ± SD. Differences
between the mice treated i.p with bestatin and the controls were
compared using the two-tailed unpaired Mest The difference between
the number of oocytes ovulated from the ovary treated with the
reagents injected into the ovarian bursa and that from the contralateral
ovary (control) was compared using the two-tailed paired r-test
Differences were considered to be significant at P < 0.05
Results
Indirect immunofluorescen.ee staining of ovarian tissues
In some 6 day old ovaries ER-BMDM1 antigen was undetectable, whereas in the others it was weakly expressed on the
cells in the theca-interstitial region (TIR cells) (data not
shown). In all 17 day old ovaries, ER-BMDM 1 antigen was
detected on the TIR cells (Figure 1) Three of the ovanes
expressed high levels of the antigen, whereas the others did
so weakly (Figure IB). In 28 day old ovaries, more than half
of them expressed high levels of the antigen on the TIR cells
(Figure ID) In 35 day old ovaries, ER-BMDM 1 antigen was
1953
K-Nakamura el aL
Figure 1. ER-BMDMl antigen expression on murine ovaries detected by indirect lmmunofluorescence staining. (A and B) A 17 day old
ovary (C and D) A 28 day old ovary. (A and C) Negative controls using the RHIg-87 antibody. (B) ER-BMDMl antigen was weakly
expressed on the theca-interstitial region (TIR) cells. (D) ER-BMDMl antigen was strongly expressed on the TIR cells. ER-BMDMl
antigen was not detected on oocytes and granulosa cells (B and D). GC = granulosa cells; TI = TIR cells. Original magnification XI20.
Table L Fluorescence intensity scores of ER-BMDMl antigen expression
on theca-interstitial region cells of munne ovaries
Age (days)
Fluorescence intensity scores
6
17
28
35
Figure 2. ER-BMDMl antigen expression on a 35 day old murine
ovary detected by indirect lmmunofluorescence staining (A) ERBMDR1 antigen was expressed at high levels on the thecainterstiOal region (TIR) cells (B) ER-BMDM1 antigen was weakly
expressed on some cells in the corpus luteum (arrows). GC =
granulosa cells; TI = TTR cells. Original magnification X120
expressed at high levels on the TTR cells (Figure 2A) and did
so weakly on some corpora lutea (Figure 2B). The antigen
was not detected on granulosa cells and oocytes in any ovary
examined. These expression profiles of ER-BMDM 1 antigen
on the TIR cells are summarized in Table I. The expression
intensity scores were significantly different among the groups
from 6 to 35 day old ovaries (P < 0.001).
Histochemical staining of ovarian tissues
The staining scores of aminopeptidase activity detected histochemically are summarized in Table II. Aminopeptidase activity was weakly detected on both the granulosa and TTR cells
1954
10
9
10
8
-
+
++
3
0
0
0
7
6
4
1
3.,b
6C
7b,d
The intensity of the antigen expression was graded in three scores based on
fluorescence intensity (- = none, + = weak; + + = intense). If
judgements differed, the lower value was selected. Differences were
analysed by the Kruskal-Wallis test followed by the Mann-Whitney test
Significant differences are denoted by *-b/) < 0.01, ^P < 0 05
Table II. Staining intensity scores of aminopeptidase activity in thecalnterstitial region (TIR) cells and granulosa cells detected by histochemistry
Staining intensity scores
Age
Caays)
6
17
28
35
TIR cells
10
9
11
9
Granulosa cells
-
+
4
0
0
0
6
5
6
0
+
4«.b
5°^
9bAc
-
+
++
4
0
0
1
6
4
4
4
O^
5f
7«
4h
The staining intensity of peptidase activity was graded in three scores based
on staining intensity (- = none; + = weak; + + = intense) and assessed
by two individuals If judgements differed, the lower value was selected.
Differences were analysed by the Kniskal-Walhs test followed by the
Mann-Whitney test
Significant differences are denoted by. tJ>^P < 0.01, bJ'P < 0.05
Bestatin enhances ovulation in mice
Ti
GC 1
B
Tt
GC
D
Figure 3. Arrunopeptidasc activity on munne ovanes detected by histochemical staining. (A and B) A 17 day old ovary. (C and D) A 35
day old ovary. (A and C) Negative control staining (B) Weak arrunopeptidase activity was detected on both granulosa and theca-interstitial
region (TTR) cells (D) High levels of aminopeptidase activity were detected on both granulosa and TTR cells. GC = granulosa cells,
TI = TIR cells. Original magnification XI20
in about half of the 6 day old ovanes, whereas it was
undetectable in the other half (Table II). In all of the 17 and
28 day old ovanes, aminopeptidase activity was detected on
both granulosa and TIR cells (Figure 3B). The staining intensity
was high in about half of the ovanes (Table H) TIR cells
expressed high levels of aminopeptidase activity in almost all
ovaries of 35 day old mice (Table II and Figure 3D). Granulosa
cells expressed high levels of aminopeptidase activity in half
of them (Table H).
In both granulosa and TIR cells, the staining intensity scores
of aminopeptidase activity were significantly different among
the groups from 6 to 35 day old ovaries (P < 0.001 and <
0.01; Table II).
The effect of bestatin on the numbers of ovulated oocytes
In mice that were treated l.p. with bestatin, the numbers of
ovulated oocytes per animal were significantly higher than
those of the control group [47.00 ± 18.13 (n = 26) versus
35.90 ± 10.14 (n = 28), P < 0.01].
The numbers of ovulated oocytes per oviduct treated with
bestatin (2 mg/ml) via the ovanan bursa 24 h before PMSG
administration were significantly greater than those of the
controls [23 70 ± 9.61 versus 17 10 ± 5 83 (n = 25), P <
0 01]. The stereoisomer had no effect, with the numbers of
oocytes from both treated and control bursae being similar
[18.20 ± 5.42 versus 18 50 ± 5 33 (n = 21)] When bestatin
(2 mg/ml) was injected just before HCG administration, the
number of ovulated oocytes per oviduct did not differ from
that of the controls [16.10 ± 896 versus 15 80 ± 6.48
(n = 17)].
Discussion
ER-BMDM1 antibody was initially raised against bone
marrow-denved mouse macrophages stimulated with macrophage colony-stimulating factor (Leenen et al., 1992). This
antibody recognized a cell membrane-bound 160 kDa protein,
and the purified antigen showed an aminopeptidase activity
compatible with AP-N. It is considered that ER-BMDM1
antigen represents the mouse homologue of human AP-N/
cluster of differentiation (CD) 13 antigen (Look et al., 1989;
Leenen et al., 1992). In this study, ER-BMDM1 antigen was
detected by lmmunohistochemical staining on murine TIR
cells but not on granulosa cells Aminopeptidase activity
compatible with AP-N was detected on TIR cells by histochemical staining Therefore, we considered that murine ILK cells
expressed biologically active AP-N.
Aminopeptidases are generally zinc metalloenzymes and
hydrolyse peptide bonds near the N-terminal end of polypeptides (Sanderink et al., 1988). They are present in many
human tissues and body fluids, and are thought to be involved
in the metabolism of proteins, as well as various peptide
hormones. Several aminopeptidases, such as AP-N, aminopeptidase A (EC 3 4 117), dipeptidyl peptidase IV and serine
di/tripeptidases, are membrane bound and have extracellular
active sites (Razak and Newland, 1992; Shipp and Look,
1993). We reported that AP-N is expressed on theca intema
cells dunng folhculogenesis in the human ovary (Fujiwara
et al., 1992a). AP-N is present on the cell surface and
can extracellularly metabolize biologically active peptides,
including enkephalin, angiotensins I and II, somatostatin and
others, by removing an N-terminal amino acid residue
(Sandennk et al, 1988, Ward et ai, 1990). A large number
1955
K.Nakamura et aL
of biologically active peptides may be involved in folliculogenesis by autocrine and/or paracrine mechanisms. We proposed that peptidases on the cell surface of human thecal cells
are involved in regulating the extracellular peptide concentration (Fujiwara et al, 1992a). As a component of target cells,
the peptidases may modulate interactions between peptides
and their specific receptors just outside the cell membrane at
the cellular level. As members of the follicular unit, thecal
cells could regulate the peptide concentration within the
growing follicle or the concentration among the follicles,
including adjacent immature follicles, at the tissue level. Thus,
we proposed that AP-N on human thecal cells serves as an
intrafollicular and interfollicular regulator.
In this study, an AP-N-like aminopeptidase activity was also
detected on murine granulosa cells by histochemistry, although
we could not determine whether the peptidase was present on
the cell surface or in the cytoplasm of the granulosa cells.
Synthetic substrates can also be hydrolysed by some other
aminopeptidases (Sanderink et al, 1988). Because immunoreactive AP-N was not expressed on granulosa cells, the
positive staining on granulosa cells by histochemistry indicated
an aminopeptidase(s) other than AP-N on these cells. There is a
similar discrepancy in the expression between immunoreactive
AP-N and histochemical aminopeptidase activity in human
endometrium. We reported that immunoreactive AP-N is
expressed on endometnal stromal cells, but not on endometrial
glandular cells, and proposed that AP-N is a cell-surface
marker of endometrial stromal cells (Imai et al., 1992).
However, histochemical staining using the same synthetic
substrate revealed that endometnal glandular cells expressed
higher levels of aminopeptidase activity than endometrial
stromal cells. This indicated that aminopeptidase(s) other
than AP-N are located on human endometrial glandular cells
(unpublished observation). In monolayer cultures of porcine
granulosa cells, we found an AP-N-like activity on the cell
surface, the inhibition of which increased the production of
progesterone and oestradiol by these cells when stimulated with
gonadotrophins (Tachibana et al., 1996). Therefore, murine
granulosa cells may also have aminopeptidase activity on
their surface.
We investigated the physiological role of membrane-bound
aminopeptidase(s) expressed in munne TTR cells and possibly
in granulosa cells in folliculogenesis in vivo. We systematically
administered the aminopeptidase inhibitor, bestatin, into the
pentoneal cavity of immature mice treated with gonadotrophins. Bestatin is a low molecular weight dipeptide isolated
by Umezawa et aL (1976), which binds to cell surfaces and
inhibits cell-surface aminopeptidases (Rich et al, 1984). It
also inhibits aminopeptidase-B and leucine aminopeptidase
(Umezawa et al., 1976). Mice treated with bestatin had more
ovulated oocytes than control animals. This suggested that
cell-surface aminopeptidases are involved in follicular growth
and/or ovulation. To confirm the direct effect of bestatin on
the ovary, we locally administered bestatin into ovarian bursa.
Bestatin given through ovarian bursa 24 h before PMSG
injection also significantly increased the number of ovulated
oocytes, suggesting a direct effect of bestatin on the ovary.
On the other hand, a similar procedure just before HCG
1956
injection had no significant effect. Thus we considered that the
inhibition of extracellular aminopeptidase activity by bestatin
facilitates follicular growth more than the ovulation phenomenon itself. However, further investigations may be required
to clarify whether or not bestatin influences only follicular
growth. This effect of bestatin on follicular growth/ovulation
in vivo is compatible with our observation of its stimulatory
effect on steroid production by porcine cultured granulosa
cells incubated with gonadotrophin in vitro.
Serine proteases such as plasminogen activators are considered to be important enzymes for matrix remodelling; they
metabolize the extracellular matrix during follicular growth
and ovulation (Bicsak et al, 1989; Tsafriri et al, 1989). An
intrabursal injection of inhibitors of serine protease inhibits
rat follicular development (Pellicer et al, 1988), and the
injection of antibodies against tissue type plasminogen activator
suppresses the ovulation rate (Tsafriri et al, 1989). Matrix
metalloproteinases are also reportedly involved in follicular
growth and ovulation. The synthetic inhibitor of metalloproteinase(s), SC 44463, inhibits ovulation in the perfused rat
ovary (Butler et al, 1991). In contrast to these proteases, the
inhibition of membrane-bound aminopeptidases by bestatin
stimulated follicular growth/ovulation. This indicated that
the mechanism affecting the follicular growth/ovulation of
membrane-bound aminopeptidases differs from that of serine
proteases. Membrane-bound aminopeptidases contribute to
follicular growth/ovulation probably by regulating extracellular
small peptide concentrations rather than by metabolizing extracellular matrices that have a large molecular mass. Thus, they
may constitute a new category of enzymes that regulate
follicular growth/ovulation. As far as we know, this study is
the first to show the possible involvement of membrane-bound
aminopeptidase(s) in follicular growth/ovulation in vivo.
In the murine theca-interstitial region there are tissue macrophages which probably express AP-N on their cell surface
(Leenen et al, 1992). The inhibition of AP-N expressed on
munne macrophages by bestatin reportedly produces macrophage activation (Schorlemmer et al, 1983; Talmadge et al,
1986). Therefore, die stimulatory effect of bestatin on follicles
may be partially caused by activation of the immune cells.
Because bestatin has the biological activity of an lmmunomodulator, clinical application to drug therapy has been investigated (Math6 et al, 1986). At present, it is applied clinically
as an oral immunostimulant for patients with leukaemia in
Japan, and few side-effects have been reported. Although the
precise mechanism(s) of the effect of bestatin on murine
follicular growth/ovulation remains unknown, the potential for
clinical use should be noted, especially in infertile patients
who respond poorly to exogenous gonadotrophin therapy for
follicular growth/ovulation induction.
Acknowledgements
We are grateful to Drs Keiko Takabatake and Takao Himeno for
technical advice. We also thank Mr Daniel Mrozek for reading the
manuscript. TTiis work was supported in part by Grants-in-Aid for
Scientific Research (nos. 05671364, 05454446 and 06671646) and
by the Naito Foundation
Bestatin enhances ovulatlon in mice
References
Bicsak, T A., Michelson, D S and Hsueh, A J W (1989) Rat granulosa ceUs
produce a novel trypsin-like protease in response to gonadotropin treatment
Biochem. Biophys Res Common., 96, 1732-1736
Butler, TA., Zhu, C , Mueller, R.A et al. (1991) Inhibition of ovulauon in
the perfused rat ovary by the synthetic collagenase inhjbitor SC 44463
BioL Reprod, 44, 1183-1188.
Fujiwara, H., Maeda, M , Imai, K. et al (1992a) Differential expressions of
aminopepudase N on human granulosa cells and theca cells J Clin.
Endocnnol Metab , 74. 91-95
Fujiwara, H., Maeda, M., Imai, K. et al (1992b) Human luteal cells express
dipepudyl peptidase IV on the cell surface J Clin. Endocnnol Metab,
75, 1352-1357
Fujiwara, H , Maeda, M , Ueda, M et al (1993) A differentiation-related
molecule on the cell surface of human granulosa cells J Clin. Endocnnol
Metab., 76, 956-961.
Fujiwara, H , Ueda, M., Fukuoka, M. et al. (1994) A new monoclonal antibody
(POG-1) detects a differentiation antigen of porcine granulosa and thecal
cells and indicates heterogeneity of theca-stromal cells Endocnnology, 134,
1132-1138
Goto, Y, Noda, Y, Nonmoto, K. et al (1992) Pregnancy achieved by
transferring blastocysts into endometnal stroma in mice Hum. Reprod., 7,
890-893.
HarteL S , Gossrau, R , Hansld, C and Reutter, W (1988) Dipepndyl peptidase
(DPP) IV in rat organs Comparison of lmmunohistochemistry and activity
histochemistry Histochemutry, 89, 151-161
Hsueh, A J W , Bicsak, TA., Jia, X-C et al (1989) Granulosa cells as
hormone targets the role of biologically active follicle-stimulating hormone
in reproduction Recent Prog Harm. Res , 45, 209—277
Imai, K., Maeda, M , Fujiwara, H et al. (1992) Human endometnal stromal
cells and decidual cells express cluster of differentiation (CD) 13 antigen/
aminopepndase N and CD10 antigen/neutral endopepodase BioL Reprod,
46, 328-334.
Kenny, AJ , O'hare, M J and Gusterson, B A (1989) Cell-surface peptidases
as modulators of growth and differentiation Lancet, il, 785-787
Leenen, PJ M., Mehs, M , Kraal, G., Hoogeveen, A.T and Ewijk, W V (1992)
The monoclonal antibody ER-BMDM1 recognizes a macrophage and
dendritic cell differentiation antigen with aminopeptidase activity Eur J
Immunol, 22, 1567-1572
Look, A.T., Ashmun, R A , Shapiro, L H and Peiper, SC (1989) Human
myeloid plasma membrane glycoprotein CD 13 (gpl50) is identical to
aminopeptidase N Clin. Invest, 83, 1299-1307.
Math6, G, Umezawa, H, Misset, J L et al. (1986) Immunomodulating
properties of bestatin m cancer patients A phase n trial Biomed.
Pharmacother, 40, 379-382
Natsuyama, S , Noda, Y, Yamashita, M., Nagahama, Y and Mon, T (1993)
Superoxide dismutase and thioredoxin restore defective p34 a)c2 kinase
activation in mouse two-cell block Biochim. Biophys Ada, 1176, 90-94
Pellicer, A., Lightman, A, Anza, A et al (1988) Follicular development is
impaired by inhibitors of senne proteases in the rat. Am. J ObsteL Gynecol,
158, 670-676
Razak, K and Newland, A C (1992) The significance of aminopeptidases
and haematopoietic cell differentiation Blood Rev, 6, 243-250
Rich, D H., Moon, B J and Harbeson, S (1984) Inhibition of aminopeptidase
by amastann and bestatin derivatives Effect of inhibitor structure on slow
binding processes J Med Chem., 27, 423-^»29
Sanderink, G -J, Artur, Y. and Siest, G. (1988) Human aminopeptidases a
review of the literature. / Clin. Chem. Clin. Biochem., 26, 795-807
Schoriemmer, H U., Bosslet, K. and Sedlacek, HH. (1983) Ability of the
immunomodulating dipepude bestatin to activate cytotoxic mononuclear
phagocytes Cancer Res , 43, 4148-4153
Shipp, MA and Look, A T (1993) Hematopoietic differentiation antigens
that are membrane-associated enzymes cutting is the key1 Blood, 82,
1052-1070
Suda, H., Aoyagi, T., Takeuchi, T and Umezawa, H (1976) Inhibition of
aminopepudase B and leucvne aminopeptidase by bestatin and its
stereoisomer Arch Biochem. Biophys., 177, 196-200
Tachibana, T, Fujiwara, H , Suginami, H et al (1996) An aminopeptidase
inhibitor, bestatin, enhances progesterone and oestradiol secretion by porcine
granulosa cells stimulated with follicle stimulating hormone in vitro Hum.
Reprod, 11, 497-502
Talmadge, J E , Lenz, B F, Pennington, R. et al (1986) Immunomodulatory
and therapeutic properties of bestatin in mice Cancer Res , 46, 4505-4510
Tsafrin, A., Bicsak, T.A., Cajander, S3 , Ny, T and Hsueh, A J W . (1989)
Suppression of ovulation rate by antibodies to tissue-type plasminogen
activator and a r antiplasmm Endocnnology, 124, 415-421
Umezawa, H., Aoyagi, T., Suda, H , Hamada, M and Takeuchi, T (1976)
Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes J
Antibiot, 29, 97-99
Ward, PJE , Benter, I F , Dick, L and Wilk, S (1990) Metabolism of vasoactive
pepndes by plasma and purified renal aminopepudase M Biochem.
Pharmacol, 40, 1725-1732
Received on Apnl 10, 1996, accepted on June 14, 1996
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