Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2000-01-07
2003-07-15
Reynolds, Deborah J. (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S325000, C800S008000, C800S021000
Reexamination Certificate
active
06593139
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to the activation of a mammalian oocyte by a coordinated activity of one or more heat-sensitive constituents and at least one heat-stable constituent of a spermatozoon submembrane perinuclear matrix.
For fertilization to be successful, sperm and egg components must mutually initiate the network of events required for full embryonic development to produce normal young. The subset of these events that immediately follow sperm-egg fusion and that leads up to the mingling of sperm and egg chromosomes is collectively termed oocyte activation [R. Yanagimachi in The Physiology of Reproduction, Second Ed., New York, Raven Press, pp. 189-317 (1994)]. The interplay between fertilizing sperm and egg factors that initiate oocyte activation is still unclear although, often by analogy with other systems, several components of potential signaling pathways have been identified within mature, metaphase-arrested oocytes (i.e., of the type encountered by a fertilizing spermatozoon.) Experimental evidence supports G protein mediated pathways in the eggs of both invertebrates [F. M. Shilling, et al.,
Dev. Biol
166, 34-58 (1994)] and vertebrates [C. J. Williams, et al.,
Dev. Biol
. 15, 288-296 (1992)] and of phospholipase C-mediated signal transduction, also in the case of both invertebrates [D. J. Carroll, et al.,
J. Cell Biol
. 138, 1303-1311 (1997)] and vertebrates [G. Dupont, et al., Biochem J. 316, 583-591 (1996)]. The breadth of this potential signaling repertoire may reflect functional redundancy with multiple pathways each capable of eliciting full activation [G. Dupont, et al., (1996)]. It is likely that Ca
2+
mobilization is pivotal to at least one of these pathways [M. J. Berridge,
Nature
361, 315-325 (1993)].
The mechanism by which oocyte activation is initiated is broadly described in two models: 1) transmembrane signaling transduction across the oolemma upon sperm-egg binding/fusion, and 2) the introduction of a sperm soluble factor that initiates activation within the ooplasm [B. Dale, et al.,
Experientia
41, 1068-1070 (1985); K.
Swann, Development
110, 1295-1302 (1990); and K. R. Foltz and F. M. Shilling,
Zygote
1, 276-279 (1993)]. The models are not mutually exclusive. The first relies in large part on the observation that oocytes from vertebrates and nonvertebrates contain signal-transducing components that, in other systems described above, respond to altered states in transmembrane receptors. So far, few “candidate receptors” have been identified, perhaps the strongest belonging to the RGD-sensitive integrins resident on the plasma membrane of Xenopus and mouse eggs [E. A. C. Almeida, et al.,
Cell
81, 1095-1104 (1995); and Y. Iwao and T. Fujimura,
Dev. Biol
. 177, 558-567 (1996)]. Recently, it has been shown that when mouse spermatozoa are microinjected directly into the ooplasm, circumventing gamete surface-surface interactions, full activation nevertheless occurs, with development of resulting embyros to term after transfer [Y. Kimura and R. Yanagimachi,
Biol. Reprod
. 52, 709-720 (1995); S. Kuretake, et al.,
Biol. Reprod
. 55, 789-795 (1996); and Y. Kimura, et al.,
Biol. Reprod
. 58, 1407-1415 (1998)]. In particular, it has been shown that, when microinjected into mouse oocytes, demembranated sperm heads are capable of eliciting full activation and embryo development to term [S. Kuretake, et al., (1996); and Y. Kimura, et al., (1998)]. This activating function resides in the sperm head, not the tail, and that it appears during spermiogenesis in the mouse [Y. Kimura, et al., (1998)]. These findings are consistent with the second hypothesis, that a sperm factor can initiate activation by interacting directly with the ooplasm. It has been suggested that sperm cytosolic components from a wide variety of mammalian species, including the mouse, hamster, monkey, rabbit, and human can play such a role [K. Swann, (1990); S. L. Stice and J. M. Robl,
Mol. Reprod. Dev
. 25, 272-280 (1990); S. T. Homa and K. Swann,
Hum. Reprod
. 9, 2356-2361, (1994); L. Meng and D. P. Wolf,
Hum. Reprod
12, 1062-1068 (1997); and K. Swann and F. A. Lai,
Bioessays
19, 371-378 (1997)]. The sperm components at the center of several previous studies can be readily liberated by brief sonication or simple freeze-thawing, although in some cases their ability to activate is inferred from the similarity between induced intracellular Ca
2+
mobilization and that observed at fertilization. Unfortunately, this is potentially misleading for two reasons: 1) it is possible that not all normal activation pathways require or exhibit Ca
2+
mobilization [R. M. Moses and D. Kline,
Mol. Reprod. Dev
. 41, 264-273 (1995)] and, conversely, 2) factors may be identified that can cause Ca
2+
mobilization in oocytes but play no role in normal fertilization or embryonic development.
Regardless of the mechanism(s) of oocyte activation, it is known that there are instances in which human and/or other mammal spermatozoa are not capable of activating oocytes. Therefore, there is still a need to identify constituents of spermatozoa that lead to full oocyte activation, sufficient for the birth of live young.
SUMMARY OF THE INVENTION
The invention provides a method of identifying certain properties of sperm-borne oocyte-activating factor(s) (SOAF) responsible for triggering full oocyte activation. It was predicted that to account for SOAF activity during oocyte activation by a demembranated spermatozoon head, an apparently detergent-insoluble sperm component with the properties of SOAF would have to be responsive to ooplasmic factors during fertilization. By the method of the invention, such a sperm component has been identified. To demonstrate the properties of this sperm component, the method of the invention includes in vitro activation of oocytes by a microinsertion-based “trans-complementation” assay, i.e., the failure of a heat-inactivated demembranated mature spermatozoon head to activate an egg is rescued by coinsertion of a soluble factor isolated from a non-heat-inactivated demembranated spermatozoon head under reducing conditions. Thus, by the method of the invention, mature spermatozoa are demembranated under conditions that provide demembranated spermatozoa heads comprising nuclei and matrix-bound perinuclear material that retain oocyte activation activity if injected into the ooplasm of oocytes. A soluble factor (SOAF
s
) is extracted from an aliquot of the demembranated spermatozoa heads by incubation in a physiological medium containing a reducing agent such as, but not limited to, dithiothreitol (e.g., about 15 millimolar, mM) or reduced glutathione (e.g., about 10 mM) at about 0° C. to about 37° C. for about 30 minutes. Another aliquot of the demembranated spermatozoa heads is heat-treated in a physiological medium, preferably under reducing conditions, at a temperature sufficient to abolish oocyte activation activity (e.g., about 44° C. to about 100° C.). Co-insertion of an inactivated demembranated spermatozoon head and an aliquot of the soluble factor into an unfertilized oocyte, results in activation of the oocyte. Thus, SOAF
s
“rescues” the activity of the inactivated demembranated spermatozoon head to activate an oocyte.
A feature of the invention is the discovery that SOAF
s
is trypsin-sensitive and is liberated from the matrix-bound perinuclear material of the demembranated spermatozoa in a temperature-dependent manner. Moreover, the ability of the demembranated spermatozoa to activate oocytes is also temperature-dependent and is inversely correlated with the liberation of SOAF
s
. The activity of SOAF
s
to “rescue” heat-inactivated demembranated spermatozoa heads in oocyte activation is abolished by heat-treatment at about 44° C. to about 100° C. Without being bound by theory, it is believed that the sperm-borne oocyte-activation factor identified by the invention is a matrix-bo
Perry Anthony C. F.
Yanagimachi Ryuzo
Day Jones
Paras, Jr. Peter
Reynolds Deborah J.
University of Hawai'i
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