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JEZ 826
Cumulus Cell Dispersal From Murine Oocytes by
an Epididymal Guanidinobenzoatase
Department of Biology, University of Alabama at Birmingham,
Birmingham, Alabama 35294
Department of Biology, Talladega College, Talladega, Alabama 35160
Guanidinobenzoatase (GB), a serine proteinase with a molecular weight of 71,000,
is found both free in the epididymal fluids of the mouse and bound to the sperm surface. Microgram quantities of the enzyme, purified from epididymal fluid, will completely disperse follicle
cells from freshly ovulated ooctyes after 15 min of incubation. Purified GB exhibits no hyaluronidase activity as determined by the acid albumin assay. The ability of GB to disperse follicle
cells is blocked by a proteinase inhibitor endogenous to the male reproductive tract. The inhibitor
has no effect on bovine testicular hyaluronidase. Although the function of GB has not been defined, the observations presented here indicate that it may play a role in cumulus matrix penetration during fertilization. J. Exp. Zool. 279:185–188, 1997. © 1997 Wiley-Liss, Inc.
The ovulated mouse oocyte is covered by two
distinct layers, the cumulus oophorus and the
zona pellucida. Fertilization requires that the
sperm penetrate both layers. It is generally believed that passage through these layers is, at
least in part, an enzymatic process involving hyaluronidase for penetration of the cumulus layer
and acrosin for the zona (Yanagimachi, ’88). The
cumulus layer is composed of approximately 3,000
cells (Lin et al., ’94) embedded in a matrix of hyaluronic acid and protein (Talbot, ’85). Ultrastructural analysis of the matrix reveals a combination
of granules and filaments. The filaments are sensitive to hyaluronidase and the granules to trypsin
(Talbot and DiCarlantonio ’84). These observations
suggest that a protease, in addition to the more
commonly implicated hyaluronidase, could be involved in cumulus matrix digestion during sperm
penetration. Data presented here indicate that a
serine proteinase, (GB), isolated from epididymal
fluids (though also found on sperm, Benau and
Storey, 1987; Barksdale et al., 1997) is able to disperse follicle cells from recently ovulated oocytes.
Seminal vesicle inhibitor (SVI) isolation
and assay
The inhibitor was isolated from seminal vesicle
tissue as previously described (Poirier and Jackson, ’81). This includes gel filtration (G-75 Sephadex), affinity chromatography with trypsin as the
ligand, and ion exchange chromatography (SPSephadex C-25). The purified material was dialyzed against PBS and stored at –20°C until used.
Proteinase inhibitor activity was measured
against porcine pancreatic trypsin using N-benzoyl-DL-arginine-P-nitroanilide (BAPNA) as the
substrate in Ca+2-free triethanolamine hydrochloride buffer, pH 7.8, 0.2M (Fritz et al., ’74). Each
3-ml assay contained 6.0 µg trypsin, 0.77 mM
BAPNA, and various amounts of inhibitor. An inhibitor unit (IU) is defined as the amount of inhibitor that reduces the hydrolysis of BAPNA by
1 µmol/min.
Isolation of guanidinobenzoatase
The caudae epididymides and ducti deferens
were removed and placed in PBS, pH 7.2. Sperm
were stripped from the ducti. The caudal epididymides were punctured and the sperm were allowed to swim free. The sperm suspensions were
Random bred mice 6–12 weeks of age were used
throughout the study. These animals were subjected to controlled lighting (16 L:8D) and constant temperature (22 ± 2°C) and had free access
to food and water.
*Correspondence to: Gary R. Poirier, Dept. of Biology, University
of Alabama at Birmingham, University Station, Birmingham, AL
Received 18 December 1996; Revision accepted 21 April 1997.
then centrifuged and the supernatants contained
GB activity.
GB was purified in two steps using molecular
sieving (Sephadex G-75) and affinity chromatography with agmatine as the ligand (Barksdale et
al., ’97).
Guanidinobenzoatase assay
Guanidinobenzoatase activity was measured
fluorimetrically using 4-methylumbelliferyl-pguanidinobenzoate (MUGB) as the substrate. The
typical assay contained 0.1 to 0.5 ml of the enzyme (GB) and PBS to a total of 2 ml. Ten microliters of MUGB (2 mg/ml in dimethylsulfoxide)
was added, final concentration of 26 µM, and the
hydrolysis was immediately monitored (excitation
wavelength of 360 nm and an emission wavelength of 450 nm) for 5 min at room temperature
in a Sequoia-Turner Model 450 Fluorimeter
equipped with a chart recorder. One enzyme unit
(U) corresponds to the formation of 1,000 ng of
product (4-methylumbelliferone) per min. One inhibitor unit (IU) inhibits the formation of product
by 1,000 ng per min.
Hyaluronidase assay
The assays were performed according to procedures suggested by Dorfman (1955). One-half milliliter of GB solutions was mixed with 0.5 ml of
enzyme buffer (0.02M phosphate buffer, 0.45%
NaCl, 0.01% BSA, pH 7.0) followed by the addition of 1 ml of substrate buffer (0.3 M KH2PO4/
Na2HPO4, pH 5.3) containing 0.5 mg of hyaluronic
acid. The mixture was incubated for 45 min at
37°C. Turbidity was then developed by adding 5
ml of an acid albumin solution (1 mg/ml albumin
in Na acetate buffer, 0.05M pH 3.7). The OD (600
nm) was determined 5 min after the addition of
the acid albumin. Bovine testicular hyaluronidase
(Sigma Chemical Co. St. Louis, MO) containing
0.3 U/µg was used to establish standard curves.
cell dispersal through an inverted microscope. Follicle cells were considered dispersed when the oocytes were clearly visible.
GB, isolated from the epididymal fluids, showed
a molecular weight band of 71,000 on a 4–20%
gradient SDS polyacrylamide gel (Fig. 1). The
preparation had a specific activity of 1.6 U/mg.
Under the conditions of the assay, GB effectively
dispersed follicle cells after a 15-min incubation
(Fig. 2, Table 1). Bovine testicular hyaluronidase
(100 µg/ml) was used as a positive control and
PBS as a negative control (Table 1). SVI is able
to block the ability of GB preparations to disperse
follicle cells but had no effect on hyaluronidase.
GB has no detectable hyaluronidase activity and
SVI does not inhibit bovine testicular hyaluronidase in the acid albumin assay (Fig. 3). Hyaluronidase, from 0.1 to 1.0 mg/ml, does not
hydrolyse MUGB (data not shown).
GB is a serine proteinase whose presence on
sperm and in seminal fluids was first described
by Steven and Al-Ahmad (’83). GB has been implicated in tumor metatasis, cell migration, and
tissue remodeling (Poustis-Delpont et al., ’92).
Benau and Storey (’87) characterized the enzyme
on murine sperm and considered it to be a trypsin
inhibitor sensitive site, which participated in zona
binding. Recently the soluble form, derived from
Murine ovulated oocytes and
cumulus dispersal
Mature female mice were superovulated by IP
injections of 10 IU of pregnant mare’s serum
(PMS) gonadotropin followed 48 hr later by 10 IU
of human chorionic gonadotropin (hCG). Ovulated
eggs, in cumulus masses, were recovered from oviducts approximately 16 hr after the hCG injection. The masses were washed twice in PBS and
transferred to 50 µl of test solution in wells of 96well microtiter plates. The plates were then incubated for 15 min at 37°C and observed for follicle
Fig. 1. SDS-PAGE (4 to 20% gradient gel) of ~ 4 µg of GB
isolated from an epididymal sperm supernatant (lane b). Molecular weight standards are in lane a.
TABLE 1. Cumulus dispersal summary1
Components tested
Hdase + SVI
Number of cumulus free oocytes after a 15 min incubation/total number of oocytes. GB, guanidinobenzoatase, used at 65/µg/ml, ~1.6 U/
mg. SVI, proteinase inhibitor of seminal vesicle origin, used at 200
mIU/ml, ~50 µg/ml. Hdase, bovine testicular hyaluronidase, used at
100 µg/ml, 0.3 U/µg.
epididymal fluids, has been characterized (Barksdale et al., ’97). Whole zonae, biologicaly active
ZP3, and proteinase inhibitors, including SVI,
block its ability to hydrolyse MUGB (Barksdale
et al., ’97). These data support the contention that
the trypsin inhibitor sensitive site functions in
zona binding.
Recently mouse PH-20, localized on the sperm
head, was shown to have hyaluronidase activity
(Lin et al., ’94). Purified GB was unable to hydrolyse hyaluronic acid and hyaluronidase did not
hydrolyse MUGB. GB is inhibited by SVI while
bovine testicular hyaluronidase is not. Even
though the molecular weight estimate (71,000) for
GB is only slightly higher than the 68,000 suggested for murine glycosylphosphatidylinositollinked hyaluronidase (Thaler and Cardullo, ’95),
it appears that GB is distinct from hyaluronidase.
The data presented here indicate that GB is able
to disperse cumulus cells, presumably by matrix
hydrolysis. Since the matrix is made of protein
granules as well as hyaluronic acid filaments it
seems appropriate that a fertilizing sperm could
use both enzyme types, perhaps synergistically,
Fig. 3. Effects of adding increasing amounts of purified
SVI (Z) on the ability of 20 µg of bovine testicular hyaluronidase to hydrolyse hyaluronic acid. Failure of various
amounts of purified GB (5) to hydrolyse hyaluronic acid. Standard curve using bovine testicular hyaluronidase (2) to hydrolyse hyaluronic acid.
Fig. 2. The effects of incubating a freshly ovulated egg
mass in a solution containing 65 µg/ml of purified GB. a: An
egg mass at zero time. b: The same egg mass after 15 min of
incubation at 37°C. × 550.
in matrix penetration. For an enzyme to function
in murine matrix penetration during in vitro fertilization it should be bound to the surface of
acrosome intact sperm (Lin et al., 1994). GB activity is present both on twice washed epididymal sperm and in epididymal fluids (Benau and
Storey, 1985; Barksdale et al., 1997). If the spermbound form of GB is like the soluble form then it
seems appropriate to suggest that is has a role in
matrix penetration.
The authors are grateful to Mrs. Isabell Boyd
and Mrs. Freddie Oglesby for excellent secretarial help.
Barksdale, Z., S.C. Caldwell, D.J. Aarons, S.B. Young, and
G.R. Poirier (1997) Characterization of the guanidinobenzoatase in epididymal fluids of the mouse. Mol. Reprod.
Dev., 47:204–209.
Benau, D.A., and B.T. Storey (1987) Characterization of the
mouse sperm plasma membrane zona-binding site sensitive to trypsin inhibitors. Bio. Reprod., 36:282–293.
Dorfman, A. (1955) Mucopolysaccharidases. Methods Enzymol., 1:166–173.
Fritz, H., I. Trautschold, and E. Werle (1974) Protease inhibitors. In: Methods of Enzymatic Analysis. H.U. Bergmeyer, ed. Academic Press, New York, pp. 1064–1080.
Lin, Y., K. Mahan, W.F. Lathrop, D.G. Myers, and P. Primakoff
(1994) A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell lyer surrounding the egg. J. Cell Biol., 125 :
Poirier, G.R., and J. Jackson (1981) Isolation and characterization of two proteinase inhibitors from the male reproductive tract of mice. Gamete Res., 4:555–569.
Poustis-Delpont, C., R. Descomps, P. Auberger, P. DelqueBayer, P. Sudaka, and B. Rossi (1992) Characterization and
purification of a guanidinobenzoatase: A possible marker of
human renal carcinoma. Cancer Res., 52:3622–3628.
Steven, F.S., and R.K. Al-Ahmad (1983) Evidence for an enzyme which cleaves the guanidinobenzoate moiety from active-site titrants specifically designed to inhibit and quantify
trypsin. Eur. J. Biochem., 130:335–339.
Talbot, P. (1985) Sperm penetration through oocyte investments in mammals. Am. J. Anat., 174:331–346.
Talbot, P., and C. DiCarlantonio (1984) The oocyte-cumulus
complex: Ultrastructure of the extracellular components in
hamster and mice. Gamete Res., 10:127–142.
Thaler, C.D. and R.A. Cardullo (1995) Biochemical characterization of a glycosylphosphatidylinositol-linked hyaluronidase on mouse sperm. Biochemistry, 30:7788–7795.
Yanagimachi, R. (1988) Mammalian fertilization. In: The
Physiology of Reproduction. E. Knobil and J.D. Neill, eds.
Raven Press, Ltd., New York, pp. 136–185.
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