U.S. patent application number 13/357782 was filed with the patent office on 2012-07-26 for method of regulating fertilizing ability using cyclic adp-ribose and cd38.
This patent application is currently assigned to Industrial Cooperation Foundation Chonbuk National University. Invention is credited to Byung-Ju Kim, Uh-Hyun Kim, Kwang-Hyun Park.
Application Number | 20120189607 13/357782 |
Document ID | / |
Family ID | 45524408 |
Filed Date | 2012-07-26 |
United States Patent
Application |
20120189607 |
Kind Code |
A1 |
Kim; Uh-Hyun ; et
al. |
July 26, 2012 |
METHOD OF REGULATING FERTILIZING ABILITY USING CYCLIC ADP-RIBOSE
AND CD38
Abstract
The present invention relates to a pharmaceutical composition
for promoting fertilization comprising cyclic ADP-ribose or its
derivative, CD38 and to a method of promoting fertilization by
promoting the synthesis of cyclic ADP-ribose to increase sperm
motility. Also, the present invention relates to a pharmaceutical
composition for contraception and a method for inhibiting
fertilization, which can inhibit the expression or function of
cyclic ADP-ribose to reduce sperm motility, thereby inhibiting
fertilization.
Inventors: |
Kim; Uh-Hyun; (Jeonju-si,
KR) ; Park; Kwang-Hyun; (Jeonju-si, KR) ; Kim;
Byung-Ju; (Jeonju-si, KR) |
Assignee: |
Industrial Cooperation Foundation
Chonbuk National University
Jeonju-si
KR
|
Family ID: |
45524408 |
Appl. No.: |
13/357782 |
Filed: |
January 25, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61436050 |
Jan 25, 2011 |
|
|
|
Current U.S.
Class: |
424/94.61 ;
435/200; 514/47; 536/26.13 |
Current CPC
Class: |
A61K 31/57 20130101;
A61K 31/708 20130101; A61P 15/08 20180101; A61K 31/57 20130101;
A61K 31/7076 20130101; A61K 38/47 20130101; A61K 2300/00 20130101;
A61P 15/16 20180101; A61K 31/00 20130101 |
Class at
Publication: |
424/94.61 ;
536/26.13; 514/47; 435/200 |
International
Class: |
A61K 31/7076 20060101
A61K031/7076; A61K 38/47 20060101 A61K038/47; A61P 15/16 20060101
A61P015/16; C12N 9/24 20060101 C12N009/24; C07H 19/213 20060101
C07H019/213; A61P 15/08 20060101 A61P015/08 |
Claims
1. A pharmaceutical composition for promoting fertilization
comprising cyclic ADP-ribose or its derivative.
2. A pharmaceutical composition for promoting fertilization
comprising CD38.
3. The pharmaceutical composition of claim 1, wherein cyclic
ADP-ribose is synthesized by CD38 (ADP-ribosyl cyclase).
4. The pharmaceutical composition of claim 1, wherein CD38 is
contained in prostasome.
5. A method of promoting fertilization by increasing the motility
of sperm, the method comprising a step of promoting the synthesis
of cyclic ADP-ribose.
6. The method of claim 5, wherein the cyclic ADP-ribose is
synthesized in prostasome-bound sperm.
7. The method of claim 5, wherein the method further comprises a
step of treating the sperm with progesterone.
8. The method of claim 7, wherein the treatment with the
progesterone promotes intracellular calcium release to induce a
continuous increase in calcium.
9. A pharmaceutical composition for contraception comprising an
antagonist of cyclic ADP-ribose.
10. The pharmaceutical composition of claim 9, wherein the
antagonist is 8-Br-cADPR or 8-amino-cADPR.
11. The pharmaceutical composition of claim 9, wherein the cyclic
ADP-ribose is synthesized in CD38 contained in prostasome.
12. The pharmaceutical composition of claim 9, wherein the
antagonist inhibits a pattern of a continuous increase in calcium
by progesterone.
13. A method for inhibiting fertilization comprising inhibiting
cyclic ADP-ribose.
14. The method of claim 13, wherein the inhibition is performed by
treatment with an antagonist of the cyclic ADP-ribose.
15. The pharmaceutical composition of claim 10, wherein the
antagonist inhibits a pattern of a continuous increase in calcium
by progesterone.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a pharmaceutical
composition for promoting fertilization comprising cyclic
ADP-ribose or its derivative. More specifically, the present
invention relates to a pharmaceutical composition for promoting
fertilization and a method for promoting fertilization, which can
increase sperm motility using cyclic ADP-ribose and CD38, thereby
promoting fertilization.
[0003] Also, the present invention relates to a pharmaceutical
composition for contraception comprising an antagonist of cyclic
ADP-ribose. More specifically, the present invention relates to a
pharmaceutical composition for contraception and a method for
inhibiting fertilization, which can inhibit a pattern of a
continuous increase in calcium caused by progesterone to inhibit
sperm motility, thereby inhibiting fertilization.
[0004] 2. Description of the Prior Art
[0005] Spermatozoa are produced in the testis and undergo
post-gonadal modifications in the epididymis to acquire fertilizing
ability. In epididymal plasma, high-molecular-weight proteins and
such small molecules as free carnitine convert the gametes into
competent and functional cells. Free L-carnitine is taken up from
blood plasma and concentrated in the epididymal lumen. This
epididymal secretion is beneficial for spermatozoa and is not
merely an excretory waste. Free carnitine goes through the sperm
plasma membrane by passive diffusion. Free L-carnitine is
acetylated in mature spermatozoa only. The excess acetyl-CoA from
the mitochondria is probably stored as acetyl-L-carnitine and
modulates the reserves of free CoA essential to the function of the
tricarboxylic acid cycle. This property of L-carnitine of buffering
CoA in the mitochondrial matrix is known in somatic cells but is
accentuated in male germinal cells. The relationship between the
endogenous pool of free and acetylated L-carnitine and the
percentage of progressive sperm motility indicates a more important
metabolic function. Thus, the potential of initiating sperm
motility which takes place in the epididymis is probably
independent of the carnitine system while the energy properties of
acetyl-L-carnitine is relevant in situations of "energy crisis".
The uptake of cytoplasmic free L-carnitine in mature spermatozoa
must be a protective form of mitochondrial metabolism useful to the
survival of this isolated cell.
[0006] Idiopathic asthenozoospermia, a disorder of sperm motility,
is illustrative of certain conditions in this area. It is a
post-testicular cause of infertility due to various ethiology, i.e.
congenital defects of the sperm tail, maturation defects,
immunological disorders or infection. Several drugs for treating
idiopathic asthenozoospermia, none of them completely satisfactory,
are known.
[0007] Antiestrogen drugs (such as clomiphene citrate and
tamoxifen) block sex hormones from inhibiting the Follicle
Stimulating Hormone (FSH) and the Luteinizing Hormone (LH) in the
brain. This triggers an increased release of LH and FSH, which in
turn stimulates testosterone production. Increased testosterone
level improves spermatogenesis, thus improving sperm density and
motility. However, a recent randomized, double-blind, multicenter
study of 190 couples by the World Health Organization (WHO) showed
no effect of clomiphene citrate. Tamoxifen was claimed to improve
sperm concentration but no change in motility was usually detected.
As for clomiphene, recent studies did not confirm its efficacy.
Testolactone, an aromatase inhibitor, prevents the conversion of
testosterone to estradiol. It has been tested in patients with
idiopathic oligospermia but contrasting results have raised many
doubts on its efficacy. Mesterolone is a synthetic androgen widely
used to treat idiopathic maleinfertility. A recent study sponsored
by WHO failed to show any efficacy of this drug. Thus, studies on a
method of promoting fertilizing ability by increasing sperm
motility are urgently required.
[0008] Calcium signaling in sperm is known to be released from the
calcium store present in the midpiece and plays an important role
in sperm motility, and calcium signaling by progesterone was
reported to occur through a new mechanism having no concern with
IP.sub.3 that is a general calcium signal initiation site (Fabiani
et al., Hum. Reprod, 9, 1485 (1994)). Furthermore, it is known that
calcium signaling occurring in many cells stimulates RyR, which
opens the calcium store by cyclic ADP-ribose, to release calcium
into the cytoplasm, but specific mechanisms in sperm and sperm
motility have not yet been reported (Arienti et al., Biol. Cell.
91, 51-54 (1999); Harper et al., J. Biol. Chem. 279, 46315-46325
(2004); Meszaros, Nature 364, 76-79 (1993)). However, it is known
that sperm has no endoplasmic reticulum, unlike other cells, and
SERCA that is an important calcium channel in the intracellular
calcium store is not present in sperm, and secretory pathway
Ca.sup.2+-ATPase is present in place of SERCA (Clapham, Cell 131,
1047-1058 (2007)).
[0009] CD38, a cell membrane protein, uses intracellular NAD as a
substrate to synthesize cyclic ADP-ribose (cADPR) and nicotinic
acid adenine dinucletide phosphate (NAADP), which release calcium
from intracellular calcium store into the cytoplasm, thereby
controlling the various functions of cells (Berridge et al., Nat.
Rev. Mol. Cell Biol. 4, 517-529 (2003); Lee H C, Mol. Med. 12,
317-323 (2006)). Also, it was reported that CD38 binds specifically
to CD31 present on the surface of other cells so as to perform
intracellular signaling (Deaglio et al., J Immunol. 1997;
160:395-402).
[0010] The mammalian sperm contains granules having a size of
several ten to several hundred nanometers, which are prostasomes
secreted from the prostate. The prostasomes bind specifically to
spermatozoa under weakly acidic conditions similar to the internal
conditions of the female vagina, resulting in structural or
functional changes (Ronquist and Brody, Biochim. Biophys. Acta 822,
203-218 (1985); Arienti et M al., Membr. Biol. 155, 89-94 (1997);
Publicover et al., Nat. Cell Biol. 9, 235-242 (2007); Burden et
al., Hum. Reprod. Update 12, 283-292 (2006)). The binding of
prostasomes to spermatozoa is known to increase sperm motility
(Fabiani et al., Hum. Reprod. 9, 1485-1489 (1994); Arienti et al.,
Biol. Cell 91, 51-54 (1999)), and it was reported that the
intracellular calcium content of spermatozoa bound to prostasomes
was increased (Arienti et al., Biol. Cell 91, 51-54 (1999)), but a
mechanism which is involved in calcium release by prostasomes has
not yet been known. The results of analysis of prostasomes by
proteomics techniques indicated the presence of several hundred
proteins, including CD38 that is the typical enzyme of ADP-ribosyl
cyclase (Palmerini et al., Cell Calcium 25, 291-296 (1999)), but
the functional role of the proteins or the correlation of the
proteins with other molecules has not yet been elucidated.
[0011] It has been reported that prostasomes present in sperm
contain various proteins, including CD38 (Palmerini et al., Cell
Calcium 25, 291-296 (1999)), and it was reported that a protein
group important in the signaling pathway of cyclic SDP-ribose
synthesized by CD38 is transferred into spermatozoa (Park et al.,
Science Signaling, 4, 31-41 (2011)), in which the protein group
typically includes progesterone receptor, vacuolar-type
H.sup.+-ATPase, ryanodine receptor, secretory pathway
Ca.sup.2+-ATPase, and novel ADP-ribosyl cyclase different from
CD38.
[0012] Thus, it is needed to find the relationship between
prostasomes and sperm motility and find a substance that regulates
sperm motility. Also, a new study on a method capable of regulating
fertilizing ability by regulating sperm motility based on these
findings is required.
SUMMARY OF THE INVENTION
[0013] It is an object of the present invention to provide a
composition and method of regulating fertilizing ability by
regulating sperm motility. More specifically, according to the
present invention, sperm motility can be regulated using cyclic
ADP-ribose and CD38, thereby effectively promoting fertilization or
inducing contraception as desired.
[0014] The present invention provides a pharmaceutical composition
for promoting fertilization comprising cyclic ADP-ribose or its
derivative.
[0015] The present invention provides a pharmaceutical composition
for promoting fertilization comprising CD38.
[0016] The present invention provides a method of promoting
fertilization by increasing sperm motility, the method comprising a
step of promoting the synthesis of cyclic ADP-ribose.
[0017] The present invention also provides a pharmaceutical
composition for contraception comprising an antagonist of cyclic
ADP-ribose.
[0018] The present invention also provides a method for inhibiting
fertilization comprising the expression of cyclic ADP-ribose.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows that ADP-ribosyl cyclase (hereinafter referred
to as CD38) contained in prostasome is transferred to the midpiece
of sperm to catalyze the synthesis of cyclic ADP-ribose by
progesterone in cells. In FIG. 1, A: the results of Western blot of
CD38 in sperm (lane 1) and prostasome (lane 2); B: the results of
analyzing cGDPR formation, the typical activity of intracellular
ADP-ribosyl cyclase, in prostasome-bound sperm or
prostasome-unbound sperm; C: the results of analyzing the effect of
pH on the binding of prostasome to sperm; D: results indicating
that the binding of prostasome to sperm is site-specific to the
midpiece; E: the results of examining the formation of
intracellular cyclic ADP-ribose and nicotinic acid adenine
dinucletide phosphate as a function of time in prostasome-bound
sperm or prostasome-unbound sperm.
[0020] FIG. 2 shows the results of analyzing intracellular calcium
changes in prostasome-bound sperm or prostasome-unbound sperm. In
FIG. 2, A (left): the distribution of calcium signals by
progesterone according to the locations (acrosome, midpiece and
principal piece); A (right): a schematic diagram of sperm and the
locations where calcium signals are measured; B: changes in calcium
signals in the midpiece by progesterone according to the presence
or absence of extracellular calcium in prostasome-unbound sperm; C:
changes in calcium signals in the midpiece by progesterone
according to the presence or absence of extracellular calcium in
prostasome-bound sperm; D: changes in calcium signals in the
midpiece by progesterone after pretreatment of prostasome-bound
sperm with 8-Br-cADPR that is an antagonist of cyclic
ADP-ribose.
[0021] FIG. 3 shows the results of analyzing the change in motility
induced by progesterone in prostasome-bound sperm or
prostasome-unbound sperm in comparison with a group treated with
8-Br-cADPR that is an antagonist of cyclic ADP-ribose.
[0022] FIG. 4 shows the results of analyzing the change in in vitro
fertilization with oocytes in prostasome-bound sperm or
prostasome-unbound sperm in comparison with a group treated with
8-Br-cADPR that is an antagonist of cyclic ADP-ribose (inserts:
non-fertilized oocyte (upper) and fertilized oocyte (lower)).
[0023] FIG. 5 shows the results of analyzing the change in
fertilization with oocytes in the mouse uterus in prostasome-bound
sperm or prostasome-unbound sperm. FIG. 6 shows the results of
examining the activation of
[0024] CD31 in sperm treated with CD38 at varying points of time.
When sperm was treated with CD38 at varying points of time, the
tyrosine phosphorylation of CD31 was increased with time, thus
increasing the interaction of CD31 with Src, SHP-2, GRB2, Shc and
SOS proteins. This suggests that CD38 increases the phosphorylation
of CD31 of sperm so that Src, SHP-2, GRB2, Shc and SOS which are
signaling proteins bind to CD31.
[0025] FIG. 7 shows the results of examining whether an acrosome
reaction increases when sperm was treated with purified
water-soluble CD38 (sCD38). When sperm was treated with
water-soluble CD38, the acrosome reaction was significantly
increased, and when sperm was treated with a Src inhibitor, a
Su6656 and ERK1/2 inhibitor and PD98059 which are the downstream
signaling proteins of CD31, the acrosome reaction was
inhibited.
[0026] FIG. 8 shows the results of examining changes in sperm
motility after treating sperm with purified water-soluble CD38
(sCD38). When sperm was treated with purified water-soluble CD38,
sperm motility was increased, and when sperm was treated with an
ERK1/2 inhibitor, a PD98059 and ras-GTP inhibitor and FTI-277,
sperm motility was inhibited.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention relates to a pharmaceutical
composition for promoting fertilization comprising cyclic
ADP-ribose or its derivative.
[0028] The present invention provides a pharmaceutical composition
for promoting fertilization comprising CD38.
[0029] The pharmaceutical composition according to the present
invention may additionally comprise a pharmaceutically acceptable
salt.
[0030] In the present invention, the pharmaceutically acceptable
salt means a salt which is conventionally used in the medical
field, and examples thereof include, but are not limited to,
inorganic ionic salts of calcium, potassium, sodium and magnesium,
inorganic acid salts of hydrochloric acid, nitric acid, phosphoric
acid, hydrobromic acid, iodic acid, tartaric acid and sulfuric
acid, salts of inorganic acids such as acetic acid, trifluoroacetic
acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic
acid, tartaric acid, fumaric acid, mandelic acid, propionic acid,
citric acid, lactic acid, glycolic acid, gluconic acid,
galacturonic acid, glutamic acid, glutaric acid, glucuronic acid,
aspartic acid, ascorbic acid, carbonic acid, vanilic acid,
hydroiodic acid, mandelic acid, malic acid, nitric acid, palmic
acid, panththenic acid, succinic acid and tartaric acid, salts of
sulfonic acids such as methanesulfonic acid, ethanesulfonic acid,
benzenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid
or naphthalenesulfonic acid, salts of amino acids such as glycine,
arginine and lysine, and salts of amines such as trimethylamine,
triethylamine, ammonia, pyridine and picoline. For example, the
pharmaceutically acceptable salt may be a salt of an inorganic acid
such as hydrochloric acid, or a salt of an organic acid such as
methanesulfonic acid.
[0031] The pharmaceutical composition of the present invention may
be administered orally or parenterally (e.g., intravenous,
subcutaneous, intraabdominal or topical routes). The dose of the
pharmaceutical composition of the present invention may vary
depending on the patient's weight, age, sex and physical condition,
diet, the mode of administration, the administration period or
interval, excretion rate, constitutional specificity, the property
of the formulation, the severity of the disease, etc.
[0032] For administration, the pharmaceutical composition may be
formulated into various forms. The pharmaceutical composition may
be formulated into various forms with a carrier. The carrier is a
solid, semi-solid or liquid formulation adjuvant that is nontoxic,
inert and pharmaceutically acceptable, and examples thereof include
fillers, extenders, binders, wetting agents, disintegrants,
dispersants, surfactants or diluents.
[0033] The pharmaceutical composition of the present invention may
be formulated in a unit dosage form. For example, the formulated
dosage unit may comprise 1, 2, 3 or 4 times or 1/2, 1/3 or 1/4
times the daily dose of the active compound. Preferably, the
individual dosage includes a one-time dose of the active compound,
which generally corresponds to all, 1/2, 1/3 or 1/4 of the daily
dose.
[0034] The pharmaceutical composition of the present invention may
be formulated into tablets, coated tablets, capsules, pills,
granules, suppositories, liquids, suspensions, emulsions, pastes,
ointments, gels, creams, powders or sprays. For example, for oral
administration, the pharmaceutical composition may be formulated
into solid formulations such as tablets, pills, powders, granules
or capsules, or liquid formulations such as suspensions, solutions,
emulsions or syrups. For parenteral administration, the
pharmaceutical composition may be formulated into injectable
solutions, suspensions, emulsions, freeze-dried agents or
suppositories.
[0035] The present invention relates to a method of promoting
fertilization by increasing sperm motility, the method comprising
promoting the synthesis of cyclic ADP-ribose. In the present
invention, cyclic ADP-ribose can be synthesized in protasome-bound
sperm.
[0036] For the binding of protasome to sperm, protasome and a sperm
protein may be mixed at a ratio of 5:1 to 2:1, and preferably
2:1.
[0037] The present invention may comprise treating protasome-bound
sperm with progesterone in order to promote the fertilization of
sperm. Treatment with progesterone enables sperm motility to be
increased by 2 times or more, the release of calcium in cells to
occur and calcium to be increased for a long time.
[0038] Also, the present invention may provide a pharmaceutical
composition and method for contraception, which can be used to
inhibit the expression and function of cyclic ADP-ribose to reduce
sperm motility.
[0039] More specifically, in the present invention, any material
that can inhibit the expression of cyclic ADP-ribose gene may be
used without limitation. More specifically, siRNA, antisense RNA,
snRNA, or aptamers may be used. Also, antibodies, antagonists or
neutralizing proteins may be used to inhibit the function of cyclic
ADP-ribose. More specifically, 8-Br-cADPR or 8-amino-cADPR may be
used as an antagonist for inhibiting the sperm motility of cyclic
ADP-ribose.
[0040] The present invention also provides a pharmaceutical
composition and method for contraception, which can use the
antagonist to inhibit the expression of cyclic ADP-ribose and
inhibit a continuous calcium increase pattern by progesterone,
thereby reducing fertilization possibility.
[0041] Hereinafter, the present invention will be described in
detail with reference to preferred examples. It is to be
understood, however, that these examples are provided for a better
understanding of the present invention and are not intended to
limit the scope of the present invention.
EXAMPLE 1
Isolation of Protasome
[0042] The isolation of protasome was performed using a
modification of the method of Palmerini et al. (Palmerini et al.,
Fertil. Steril. 80, 1181-1184 (2003)). The human semen was taken
and 4-fold diluted with Tris buffered saline (TBS: 30 mM Tris, pH
7.4, 130 mM NaCl), and the dilution was centrifuged at 3,000 rpm
for 30 minutes to collect the supernatant. The supernatant was
centrifuged using a high-speed centrifuge (Beckman) at
15,000.times.g for 20 minutes, and then the supernatant was
centrifuged using a ultra-high speed centrifuge at 105,000.times.g
for 2 hours, and the precipitate was collected. The collected
precipitate was resuspended in the same buffer, and a fraction
which was first eluted using Sephadex G-200 (Sigma, USA) was taken,
centrifuged and then suspended.
EXAMPLE 2
Isolation of Sperm
[0043] In order to isolate protasome-unbound sperm, ejaculated
semen was immediately 20-fold diluted with 1% BSA-containing
Bigger, Whitten, and Whittingham (BWW) medium [10 mM Hepes, 20 mM
sodium lactate, 5 mM glucose, 0.25 mM sodium pyruvate, penicillin G
(80 mg/liter), streptomycin sulfate (50 mg/liter), 95 mM NaCl, 4.8
mM KCl, 1.3 mM CaCl.sub.2, 1.2 mM KH.sub.2PO.sub.4, and 1.2 mM
MgSO.sub.4 in 25 mM NaHCO.sub.3 buffer, pH 7.4] preheated to
37.degree. C. The dilution was centrifuged at 900.times.g for 20
minutes, and the precipitate was washed twice with the same
buffer.
EXAMPLE 3
Binding of Prostasome to Sperm
[0044] The isolated sperm were diluted with a weakly alkaline
buffer or fusion buffer (150 mM NaCl, 1 mM CaCl.sub.2, 1 mM
MgCl.sub.2, 5 mM glucose with either 2 mM Hepes (pH 8.0) or 20 mM
MES (pH 5.0, fusion buffer)) containing 0.32 M sucrose. The
dilution was mixed with prostasome such that the ratio of
prostasome: sperm protein was 2:1. The mixture was allowed to react
at 37.degree. C for 15 minutes. The reaction product was
centrifuged at 600.times.g for 10 minutes, and the precipitate was
collected, resuspended in BWW buffer and used in the
experiment.
[0045] As a result, as can be seen in FIG. 1, the sperm had little
or no ADP-ribosyl cyclase (hereinafter referred to as CD38), but
the prostasome contained a large amount of CD38. The formation of
cGDPR (the typical enzymatic activity of CD38) in the
prostasome-unbound sperm and the prostasome-bound sperm was
measured, and as a result, it could be seen that the
prostasome-bound sperm had very high CD38 activity. Also, the
binding of prostasome was the highest at a pH of 5.0 similar to the
internal acidity of the female vagina and decreased toward alkaline
pH values. The prostasome was bound specifically to the midpiece of
sperm, as demonstrated by detecting CD38 using an immune staining
method. In order to examine the formation of cyclic ADP-ribose and
nicotinic acid adenine dinucletide phosphate which are typical
metabolites of CD38, the sperm to which prostasome was unbound or
bound was treated with progesterone, after which the level of each
molecule in the sperm was measured at varying points of time. As a
result, it could be seen that the synthesis of cyclic ADP-ribose
rapidly increased only in the prostasome-bound sperm within several
seconds and did not increase in other groups. Also, nicotinic acid
adenine dinucletide phosphate was not formed in any of the groups,
suggesting that the signaling of sperm by progesterone has no
connection with nicotinic acid adenine dinucletide phosphate.
EXAMPLE 4
Measurement of Calcium in Sperm Cells
[0046] In order to measure intracellular calcium release, the sperm
suspension pretreated with fluo-3 (molecular probe, USA) was
titrated in a confocal dish (SPL, Seoul, Korea) coated with
poly-L-lysine (Sigma-Aldrich, USA) and was allowed to react in a
CO.sub.2 incubator for 20 minutes to attach the cells. Calcium in
the cells was measured by a confocal microscopy system (Nikon)
using the method of Tsien et al. (Tsien et al., Nature 295, 68-71
(1982)).
[0047] As a result, in the prostasome-bound sperm, intracellular
calcium release immediately occurred by progesterone so that a
pattern of a long-lasting calcium was shown, whereas the
prostasome-unbound sperm showed an immediate calcium reaction by
progesterone, but did not a pattern of a continuous increase in
calcium. This long-lasting in calcium was calcium-dependent, but
when the prostasome-bound sperm was pretreated with 8-br-cADPR (an
antagonist of cyclic ADP-ribose) and then treated with
progesterone, the pattern of a continuous increase in calcium was
inhibited (FIG. 2). In conclusion, it can be considered that the
pattern of the continuous increase in calcium in sperm is
attributable mainly to cyclic ADP-ribose and that this molecule is
attributable to the role of CD38 transferred by the binding of
prostasome to sperm.
EXAMPLE 5
Test for Sperm Motility by Prostasome
[0048] Sperm motility was measured using a CASA (computer assisted
sperm analysis) system (IVOS, Hamilton Thorne Biosciences, USA).
The sperm suspension was titrated onto a sperm analysis slide
(2X-CEL, Hamilton Thorne Biosciences, USA) having a depth of 20
.mu.m and was automatically photographed using a 4.times.
magnification lens, followed by analysis.
[0049] As a result, the motility of the prostasome-bound sperm
increased within the error range compared to the prostasome-unbound
sperm, but was not significant. When the prostasome-bound sperm was
treated with progesterone, the motility thereof increased by about
2 times (FIG. 3). Such results could likewise be confirmed from the
results of measuring the velocity of curvilinear line (VCL) the
velocity of straight-line (VSL), the velocity of average path (VAP)
and hyperactivation motility.
EXAMPLE 6
In Vitro Fertilization Test
[0050] In vitro fertilization was performed using a modification of
the method of Ren et al. (Ren et al., Nature 413, 603-609 (2001)).
10-12-week-old mice were injected with pregnant mare serum
gonadotropin (PMSG) (5 IU; Sigma-Aldrich), and after 48 hours,
injected with human chorionic gonadotropin (hCG) (5 IU;
Sigma-Aldrich). 14 hours after the final injection, the uterine
tube was removed and perfused with IVF medium (Medicult, USA), and
the released oocytes were collected using a low-magnification
microscope. In order to collect sperm, the epididymis of male mice
was removed and cut into small pieces with scissors in the same
medium, and swum-out sperm were collected. The number of cells in
the sperm was measured, and the sperm were used in the test.
[0051] As a result, in the case of the prostasome-unbound sperm,
the ability to fertilize the mouse oocytes was reduced by about 70%
compared to the case of the prostasome-bound sperm. When the sperm
were pretreated with 8-Br-cADPR that is an antagonist of cyclic
ADP-ribose, the fertilizing ability was reduced by about 70% even
in the case of the prostasome-bound sperm. Such results suggest
that cyclic ADP-ribose which is formed by CD38 contained in
prostasome regulates the function of sperm, thereby determining the
fertilizing ability of sperm (FIG. 4).
EXAMPLE 7
Intrauterine Insemination Test
[0052] For intrauterine insemination, ovulation was induced in the
same manner as in Example 6. 5 hours after injection of hCG, the
abdominal cavity was incised so as to expose the ovary and the
uterine tube, each of prostasome-bound sperm and control sperm was
injected into the uterine tube of each animal using a catheter.
Herein, in order to facilitate the comparison of sperm motility,
the location of injection was determined to be a portion close to
the uterine cervix so as to provide a space allowing movement to
oocytes. 36 hours after the microsurgery, the mice were sacrificed,
and the uterine and the uterine tube were removed and perfused with
artificial fertilization medium. The total number of oocytes
contained in the discharged medium and the number of the cells at
the two-cell stage or older in the oocytes were counted, and
fertilization rate was calculated using the following equation:
Fertilization rate (%)=number of fertilized embryos at two-cell
stage or older/total number of oocytes.times.100
[0053] As a result, the prostasome-bound sperm showed a
fertilization rate of about 10% of the total oocytes, but in the
case of the prostasome-unbound sperm, no fertilized embryo could be
observed (FIG. 5). Such results suggest that cyclic ADP-ribose
synthesized by CD38 contained in prostasome transferred to sperm
can activate sperm motility even in vivo, thereby increasing the
fertilizing ability of sperm.
EXAMPLE 8
Test for Activation of CD31 in Sperm by CD38
[0054] In order to test the activation of CD31 in sperm, the
isolated sperm were treated with 600 ng/ml CD38 for each of 0 min,
15 min and 30 min, and then buffer was added thereto to lyse the
cells. Also, CD31 antibody was used to perform immunoprecipitation,
and Western blot was performed using each antibody in order to
examine the phosphorylation of CD31 and to examine interacting
proteins.
[0055] As a result, it was seen that the phosphorylation of CD31
increased over time and that Src, SHP-2, GRB2, Shc and SOS as
interacting proteins were involved (FIG. 6). Thus, it was believed
that signaling for activation of CD31 by stimulation of CD38 would
occur through CD31 tyrosine phosphorylation/Src/SHP-2/GRB2/Shc/SOS
proteins and that SOS would convert ras-GDP to ras-GTP to activate
ERK1/2. In addition, inhibitors of the upstream proteins were used
in order to examine whether the proteins are involved in sperm
motility and an acrosome reaction.
EXAMPLE 9
Acrosome Reaction of Sperm by CD38
[0056] To examine the acrosome reaction of sperm, ConA-FITC binding
specifically to the acrosome of sperm was used. The isolated sperm
were allowed to react with purified CD38 at 37.degree. C. for 2
hours, and then were treated for 40 min with A23187 that increases
calcium in sperm to induce the acrosome reaction of sperm. Then,
the sperm were placed on a glass slide, and the acrosome reaction
of the sperm was examined with a fluorescence microscope. As a
result, the acrosome reaction was significantly increased by CD38
and was inhibited by the Src inhibitor Su6656 and the Erk1/2
inhibitor PD98059 (FIG. 7). This suggests that CD31 is activated by
CD38 and involved in the activation of the acrosome reaction by
Erk1/2 signaling.
EXAMPLE 10
Test for Motility of Sperm by CD38
[0057] The relationship between CD31 activation by CD38 and sperm
motility was analyzed. As a result, it was seen that, when sperm
were treated with CD38, the motility of the sperm was significantly
increased (FIG. 8). Also, it was seen that, when sperm were treated
with the ERK1/2 inhibitor PD98059 and the ras-GTP inhibitor
(FTI-277) and then treated with CD38, the increase in the motility
of the sperm was inhibited. This suggests that the motility of CD31
activated by CD38 was increased by Erk1/2 signaling.
[0058] As described above, according to the present invention, the
motility of sperm can be regulated using cyclic ADP-ribose, thereby
promoting fertilization or inducing contraception as desired.
* * * * *