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a Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii 96822
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mediated signal transduction, also in the case of both invertebrates [4] and vertebrates [5]. The breadth of this potential signaling repertoire may reflect functional redundancy, with multiple pathways each capable of eliciting full activation [6, 7]; it is likely that Ca2+ mobilization is pivotal to at least one of these pathways [8]. The mechanism by which oocyte activation is initiated is broadly described in two models: 1) transmembrane signal transduction across the oolemma on sperm-egg binding/fusion and 2) the introduction of a sperm soluble factor that initiates activation within the ooplasm [9–11]. 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, respond to altered states in transmembrane receptors (see above). 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 [12, 13]. Recently, we have shown that when mouse spermatozoa are microinjected directly into the ooplasm, circumventing gamete surface-surface interactions, full activation nevertheless occurs, with development of resulting embryos to term after transfer [14–16]. This is consistent with the second hypothesis, that a sperm factor can initiate activation by interacting directly with the ooplasm. Indeed, it is suggested that sperm cytosolic components from a wide variety of mammalian species, including the mouse, hamster, rabbit, monkey, and human, can play such a role [10, 17–20]. 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 Ca2+ 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 Ca2+ mobilization [21], but conversely, 2) factors may be identified that can cause Ca2+ mobilization in oocytes but play no role in normal fertilization or embryonic development.
With this in mind, we are studying the constituents of mouse spermatozoa that lead to full activation, sufficient for the birth of live young. We have shown that when microinjected into mouse oocytes, demembranated sperm heads are capable of eliciting full activation and embryo development to term [15, 16]. We have shown that this activating function resides in the sperm head, not the tail, and that it appears during spermiogenesis in the mouse [16]. We have now investigated the properties of the sperm-borne oocyte-activating factor(s) (SOAF) responsible for triggering this full activation. We reasoned that to account for the activity, an apparently insoluble sperm component with the properties of SOAF would have to be responsive to ooplasmic factors during fertilization; we have therefore determined its sensitivity to temperature and reducing agents. On the basis of our observations, we have developed a trans-complementation assay that detects rescue of inactivated spermatozoon heads by solubilized sperm fractions. This approach allows us to dissect the contribution to oocyte activation of sperm components known to breach the oolemma at fertilization. In this way, we show that activation by demembranated spermatozoa proceeds via the coordinated action of at least two sperm head components, including a relatively insoluble, matrix SOAF (SOAFm) that can be solubilized in vitro in reducing conditions. This suggests a new model in which mammalian oocyte activation sufficient for full development is initiated via essentially insoluble (as opposed to cytosolic) sperm head components that become solubilized in response to the ooplasm when introduced into the egg at fertilization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocyte donors (B6D2F1), sperm donors (B6D2F1), and foster mothers (Swiss Webster, CD1 or ICR) were maintained within the guidelines of the Laboratory Animal Service at the University of Hawaii and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources National Research Council (DHEW publication no. [NIH] 80–23, revised in 1985). Animal-handling protocols were as reviewed and approved by the Animal Care and Use Committee at the University of Hawaii.
Preparation of Oocytes
Mature oocytes were collected from the oviducts of eCG-primed (5 IU), superovulated, 4- to 10-wk-old female B6D2F1 mice 14.5–16 h after the i.p. administration of 5 IU hCG. The cumulus cell mass was dispersed by immediate treatment in CZB-H (CZB buffered with 20 mM Hepes, pH 7.4 [14]) containing 0.1% (w:v) bovine testicular hyaluronidase (300 U/mg; ICN Biochemicals, Costa Mesa, CA) for 5–10 min at room temperature. Cumulus-free oocytes were washed four times in CZB-H and transferred to a drop of CZB under mineral oil (Squibb&Sons, Princeton, NJ) equilibrated in 5% (v:v in air) CO2 at 37°C. Data were collected exclusively from oocytes for which primary microinjection was performed within 20 h of hCG administration (the fertilizable life of ovulated mouse oocytes is more than 12 h, and delayed in vivo insemination of mice at 21–22 h post-hCG gives rise to apparently normal zygotes at control rates [22]).
Preparation of Spermatozoa and Sperm-Derived Material
Core Protocol Mouse spermatozoa were obtained by finely chopping the caudae epididymides of 1–3 freshly killed, 8- to 20-wk-old male B6D2F1 mice in 600 µl Nuclear Isolation Medium (NIM: 125 mM KCl, 2.6 mM NaCl, 7.8 mM Na2HPO4, 1.4 mM KH2PO4, 3.0 mM EDTA; pH 7.45) [15]. The pH of all incubation cocktails was in the range 7.07–7.50. Sperm suspensions were filtered through a single tissue paper (KimWipe; Kimberly-Clarke Corp., Roswell, GA) to remove tissue debris. All subsequent steps were performed at 0–4°C. The filtered sperm suspension was made 0.05–0.1% (v:v) with respect to Triton X-100 and subjected to three 5-sec bursts of sonication (60% output) from a Biosonik sonicator (Bronwill Scientific, Rochester, NY). This yielded > 99.9% decapitation and completely demembranated sperm heads [15]. Sperm fragments were pelleted for 3 min at 20 000 x g and then washed thoroughly in approximately 2 ml NIM twice at 2°C, with pelleting times of 6 and 25 min (20 000 x g). In cases in which different sperm treatments were necessary, the washed sperm suspension was divided between fresh tubes prior to the final pelleting. Pellets were typically resuspended in 100 µl NIM (giving 2–10 x 107 sperm/ml) containing, where appropriate, dithiothreitol (DTT) or reduced glutathione (GSH). An aliquot of each test suspension was removed and held on ice just prior to, and for the duration of, the incubation; this served as positive control. In most experiments, the remainder of the test suspension was incubated for 30 min at the desired temperature. In parallel, aliquots of the same freshly prepared batch of cells were inactivated by heating at or above 44°C (see Results) either for use as a negative control or with the test supernatant (see below). Hence, most experiments included 3 incubations (positive and negative controls and the test) prepared from a single batch of cells. In all experiments, a parallel, mock incubation of NIM ± DTT/GSH alone was used to generate a buffer control not exposed to spermatozoa. After incubation, spermatozoa were pelleted by spinning at 2°C for 50–80 min at 20 000 x g. The cell-free supernatant produced from test suspensions was carefully removed to a fresh tube; control supernatants were generally discarded. Pellets were resuspended either in 20 µl buffer control (positive and negative controls) or in 20 µl test supernatant. Samples were injected on the day of preparation. Resulting suspensions were added to 20 µl 20% (w:v) polyvinylpyrrolidone (PVP; average Mr 360 x 103) for injection. Where appropriate, test supernatant in the absence of heads was mixed with an equal volume of 20% (w:v) PVP prior to injection. The Core Protocol is summarized in Figure 1.
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In trypsinization experiments, soluble SOAF (SOAFs) samples (22.5 µl) were added to 10-strength trypsin solution (in NIM) to give the various final trypsin concentrations, and incubation continued at 27°C for 10 min. Reactions were then quenched on ice and protease inhibitors added (to a final concentration of 100 µM each of leupeptin, antipain, and soybean trypsin inhibitor). Positive and negative control heads (prepared by the Core Protocol and subsequently either unheated or inactivated at 48°C) were resuspended in a cocktail of 100 µM protease inhibitors plus 100 µg/ml trypsin and gave 100% and 0% activation, respectively.
For the experiments whose data are presented in Tables 3 and 4, 40 µl of a suspension of amine-modified latex beads (mean diameter 2.16 µm; Cat. no. L-0280, Sigma Chemical Co., St. Louis., MO) were washed in NIM, and the final pellet was resuspended in SOAFs or cytosolic extract and incubated on ice for 5 min. Injections of SOAFs alone were within 1 h of sample preparation. Injection volume in the absence of sperm heads was estimated from the advancement of the mercury front within the needle. Activation in the absence of sperm heads was judged to have occurred by the appearance of a second polar body and a single pronucleus.
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Spermatozoa from the golden hamster were prepared from the single epididymis of a freshly killed, sexually mature male. Spermatozoa from other species were obtained from ejaculates and processed as described in the Core Protocol section above. Sperm heads from the bull and boar were purified after a preliminary wash of ejaculates through a 0.25 M sucrose-NIM cushion. Resulting pellets corresponding to approximately half-strength ejaculate were resuspended in 10 ml NIM containing 0.2–0.4% (v:v) Triton X-100 and sonicated in eight 5-sec bursts. Sonicated spermatozoa were diluted 3-fold in ice-cold NIM, and heads were pelleted through 30 ml 75% (w:v) sucrose-NIM in 5-ml batches by spinning at 16 000 rpm for 20 min in a Sorvall (Newtown, CT) RC-5 centrifuge. Pelleted sperm heads were washed three times in NIM, resulting in a head:tail-fragment ratio of 
~10:1. Washed spermatozoa were finally resuspended in NIM-15 mM DTT, giving typical densities of 0.5–1.5 x 109 sperm/ml.
Soluble-Factor Protocol Golden hamster sperm cytosolic extracts were prepared essentially as described previously [10]. For the isolation of mouse sperm cytosolic extracts, caudae epididymides from 4 freshly killed, 12- to 20-wk-old B6D2F1 males were finely chopped in CZB-H, and the sperm were allowed to disperse before the suspension was filtered through a KimWipe. Sperm were then pelleted at room temperature for 5 min at 2000 x g and resuspended to a density of approximately 3 x 108 sperm/ml in NIM containing 1 mM DTT, 100 µM leupeptin, 100 µM antipain, and 100 µg/ml soybean trypsin inhibitor. The suspension was subjected to 4 cycles of freezing (5 min per cycle in liquid N2) and thawing (5 min per cycle at 15°C), after which sperm were pelleted at 2°C for 50 min at 20 000 x g. The resultant supernatant was carefully removed and prepared for microinjection by the addition of PVP solution as described for the Core Protocol and was used within 2 h of preparation.
Manipulation of Mouse Eggs and Embryos
Piezoelectrically actuated microinjection of sperm heads and sperm-derived material into mouse eggs has been described previously [14]. Injections were usually completed within 18–19 h post-hCG administration. Each injection session included positive and negative sperm head controls (with respective average values of 6.4 and 5.4 eggs successfully injected per experiment) and was performed with a single needle to control against interneedle variation. Data from injection sessions in which positive and negative controls did not respectively give 100% and 0% activation were discarded. (In these experiments overall, 1.2% of positive control injections failed to activate, and 1.4% of negative control injections activated; data not shown.) The injection needle tip diameter was typically 5 µm. Test solution (usually with a single sperm head) was drawn into the pipette after expulsion of a small amount of mercury into the test solution droplet. This ensured that ahead of the mercury boundary, the test solution filled the pipette and was therefore not diluted further. Since the mercury front commonly advanced by around 55 µm (the equivalent of seven mouse sperm head lengths) during injection, the equivalent of approximately 0.5 pl of the test incubation was delivered into the ooplasm when allowance is made for 1:1 dilution in PVP solution; 0.5 pl corresponds to 0.01–0.05 mouse sperm heads in the test incubation according to the Core Protocol. Sperm-injected oocytes were maintained in operation medium (CZB-H) for 2–60 min prior to transfer to CZB under mineral oil equilibrated in 5% (v:v in air) CO2 at 37°C.
Where appropriate, oocytes were artificially activated by incubation, immediately after injection, for 45–60 min in Ca2+-free CZB containing 6.7 mM SrCl2 [23, 24], under mineral oil equilibrated in 5% (v:v in air) CO2 at 37°C. After this time, eggs were washed briefly in, and transferred to, fresh CZB, and incubation was continued. Culture in vitro of embryos was in CZB for up to 4 days.
Two-cell embryos (after approximately 1 day of culture) or morulae/blastocysts (after 2.5–3.5 days of culture) were transferred to the oviducts of pregnant albino Swiss Webster or pseudopregnant albino ICR/CD1 mice that had been mated with vasectomized males of the same strain on the evening of microinjection [14]. Young born as a result of in vitro manipulation were thus characterized by their black eyes and coat color.
Collection and Processing of Data
Ova were scored at 6–10 h postinjection. Eggs that were discernibly dead were counted and discarded. The remainder were examined by light microscopy and/or aceto-orcein staining for activation evidenced by 1) the formation of two clear pronuclei (or one in cases in which no sperm head had been injected), 2) the presence of the second polar body, and 3) a change to grainy, heterogeneous cytoplasmic texture [14]. After sperm head injection, zygotes containing an odd number of pronuclei (number of pronuclei 
2 in 6.5% of cases) were scored as having activated. The data in Figure 2 gave generally good fits (residual deviance < 0.5, 3–5 degrees of freedom) to a logistic-binomial model [25], with the exception of those for 15 mM DTT (estimate of regression coefficient = 10.1, SE = 1.49). Intra-ooplasmic sperm chromatin de- and recondensation were monitored following aceto-orcein staining of injected eggs (Fig. 3).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have previously shown that sperm heads demembranated by treatment with Triton X-100 are capable of supporting embryonic development in vitro and to term following embryo transfer to a foster mother [15, 16]. As part of our ongoing effort to characterize the sperm head factor(s) responsible for this activation, we sought to establish the effect of temperature. Mouse spermatozoa prepared using the Core Protocol were resuspended in NIM containing 0, 15, or 170 mM DTT (pH 7.07–7.45) and subjected to incubation for 30 min over a range of temperatures prior to injection into metaphase II oocytes (Fig. 2). These data show an inverse correlation between temperature and the ability of sperm heads to activate within the range 15–44°C. Given that the ooplasm of metaphase II mouse oocytes represents a relatively strong intracellular reducing environment [26], we reasoned that the inclusion of reducing equivalents might also affect the ability of heads to activate, and we tested this over a range of temperatures (Fig. 2). We found that the temperature relationship was indeed apparently altered by the inclusion of the reducing agent DTT, with 15 mM DTT rendering sperm more sensitive to temperature and 170 mM DTT rendering them less so below 44°C. Physiological concentrations of the weaker reducing agent, GSH (10 mM; the concentration of GSH within unfertilized metaphase II mouse oocytes is 9–10 mM [26]), rendered sperm heads more acutely sensitive to inactivation at 37°C but otherwise mirrored results obtained with NIM alone (Fig. 2).
In all experiments, the ability of demembranated sperm heads to activate was completely abolished after treatment at or above approximately 44°C. Simultaneous injection of three 47.5°C-inactivated heads into each egg also failed to induce activation (data not shown). We investigated the fate within the metaphase II ooplasm of sperm nuclei that had been heated at 45°C (Fig. 3) or that had been subjected to 100°C for 10 min (data not shown). We found that in both cases, sperm heads had undergone decondensation and were (partially) recondensed into chromosomes within the unactivated, metaphase II oocyte. Sperm heated at 45°C had initiated this process within 2 h of injection (Fig. 3A), with the formation of chromosomes discernible after 4 h (Fig. 3B) and apparently complete within 6 h (Fig. 3C). This demonstrates that mouse sperm decondensation (in the absence of pharmacological agents) is independent of oocyte activation. In addition, it shows that sperm either do not contribute a factor that is obligate for chromatin decondensation or that the factor is extremely heat stable (and thus unlikely to be enzymic).
Heads that retained much of their ability to activate oocytes (after incubation in NIM-15 mM DTT at 27°C) were indistinguishable by electron microscopy from those completely lacking activity (after incubation in NIM at 45°C) (Fig. 4). From this and our previous work, we infer that some or all of the oocyte-activating function of spermatozoa resides in the perinuclear matrix that is resistant to treatment with Triton X-100 [15, 16]. Moreover, temperature-induced changes in the ability of sperm heads to activate are thus not due to gross structural alterations of the perinuclear matrix.
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It is not possible from these experiments to determine whether the augmented abrogation of sperm head-activating function by 15 mM DTT is a direct, negative effect (by inhibition of the factor[s] responsible) or an indirect, positive one (stimulating one or more components that subsequently inhibit or remove the activating function). However, the preservation of activating ability by 170 mM DTT (up to ~37°C) indicates the latter, since it is unlikely that relatively low, but not high, concentrations of DTT would directly abolish the activating function. This would be consistent with either the destruction of sperm-borne activating factor(s) or the liberation of SOAF from the sperm head in an active form. To investigate this further, we determined whether SOAF activity could be demonstrated in the supernatant after incubation of membrane-free sperm heads in NIM-15 mM DTT.
Temperature-Sensitive Loss of Sperm-Activating Function Was Efficiently Rescued by Trans-Complementing, Solubilized SOAF Derived from InsolubleSperm Compartments
Supernatant generated by incubation of membrane-free sperm heads in NIM-15 mM DTT over a range of temperatures was used to resuspend cells whose activity had been abolished by parallel treatment at either 100°C for 10 min or 46–49°C for 30 min. (In each experiment, complete inactivation of these sperm heads was confirmed by injection of a sample of the heads resuspended in control buffer alone.) Coinjection of inactivated heads and supernatant enabled careful control and monitoring of the volume of soluble material injected (typically 1 pl [supernatant-PVP] corresponding to 0.01–0.05 spermatozoa).
The results presented in Figure 5 show that incubation of demembranated sperm heads in NIM-15 mM DTT at temperatures of 27°C or 30°C indeed liberated an activity whose coinjection efficiently rescued the ability of 48°C-inactivated spermatozoa to induce oocyte activation (Fig. 5). We refer to the factor(s) liberated in this way as soluble SOAF (SOAFs) and to its relatively insoluble, matrix-bound progenitor(s) as SOAFm. SOAFs activity fell away in supernatants generated at higher temperatures (Fig. 5) or in the absence of DTT (data not shown) and was not detected in samples obtained above 37°C (Fig. 5). Moreover, heating SOAFs at 48°C abolished its activity in a manner that mimicked heat inactivation of whole heads (Table 1). This finding, the observation that SOAFs is liberated at temperatures at which sperm heads start to lose their activity, and the apparent origin of SOAFs from a compartment known to enter the egg at fertilization, strongly suggest that SOAFs is involved in oocyte activation and is responsible for the observed temperature sensitivity of spermatozoa in this respect.
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When heads that had been incubated at 37°C in NIM-15 mM DTT were injected, only 3.8% of eggs were activated (Fig. 2). This is consistent with loss of activity from heads due to liberation of insoluble, matrix SOAF (SOAFm) into the surrounding medium in a soluble form (the SOAFm 
SOAFs transition). However, SOAF activity was also not high in the resulting supernatant, with trans-complementation of inactivated heads in only 2 of 17 (11.8%) of injections (Fig. 5). We wished to investigate whether the absence of activity in the supernatant generated at 37°C reflected an inherent SOAFs instability at this temperature. We found that SOAFs activity generated at 27°C survived incubation for a further 30 min at 37°C, giving 100% activation when coinjected with inactivated sperm heads (Table 1). This finding, coupled with the inability of supernatants generated at 37°C, but not 27°C, to promote efficient activation, indicates SOAFs degradation in the presence but not absence of sperm heads (Table 1; Fig. 5). As part of our ongoing investigation of this phenomenon, we sought to reproduce the degradation of SOAFs generated at 27°C by trypsin proteolysis. We found that SOAFs activity was indeed abolished after treatment with trypsin (Table 1). The sensitivity of SOAFs to trypsin and heat suggests that it is proteinaceous.
The loss of activation function in 48°C-heated mouse sperm could be efficiently rescued in SOAFs samples generated by incubation of demembranated sperm from the human, pig, bull, or hamster in NIM-15 mM DTT at 27°C (data not shown). SOAFs is therefore not highly species specific. Demonstration of SOAFs has been achieved using pig sperm heads that have been highly purified, with < 5% contamination by tail fragments (data not shown). Porcine SOAFs activity resides in extracts that are typically 200–400 µg total protein/ml (that is, a maximum of approximately 0.2 pg of solubilized, sperm-derived protein is introduced per injection). Furthermore, SOAFs from the pig and mouse activated mouse oocytes sufficiently for embryonic development to the blastocyst stage and beyond, to term (Table 2). Developmental rates (to term) of oocytes activated by SOAFs or treatment with SrCl2 are similar, but low. However, analysis of oocytes injected with sperm prepared according to the Core Protocol revealed a very high level of structural abnormalities in male-derived chromosomes (data not shown). These data suggest that the low developmental rate reflects chromosomal damage resulting from sperm preparation using the Core Protocol.
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Lack of Evidence for a Direct Interaction between 48°C-Heated Heads and SOAFs in Initiating Activation
To probe further the nature of any interaction between coinjected SOAFs and inactivated sperm heads, we injected SOAFs alone or with latex beads as adjuvants (i.e., in the absence of heads). Neither treatment induced the resumption of meiosis (Table 3). However, SOAFs was capable of rescuing heat-inactivated spermatozoa that had previously been sonicated either not at all or briefly in the absence of Triton X-100 (data not shown). In this situation, sites that would have been exposed for specific SOAFs binding in Triton X-100-demembranated cells presumably remain largely occluded. If so, any SOAFs complexes forming on these cells would presumably be nonspecific or form intra-ooplasmically.
The potential for interaction between SOAFs and inactivated heads was probed further by injecting SOAFs and inactivated heads into the same egg but at separate times. Oocytes were first injected with inactivated heads (in NIM-15 mM DTT) and, after incubation at 37°C for 2 or 4 h, subsequently injected with SOAFs. This resulted in activation of 11 of 14 (79%) of oocytes, although negative controls in which the secondary injection was with SOAFs that had been inactivated by heating at 48°C for 30 min failed to activate (0 of 5). This shows that SOAFs possesses activity in the absence of inactivated heads (i.e., they do not merely stabilize SOAFs) and that exposure of the ooplasm to heads and SOAFs does not have to be simultaneous for activation to occur. Given the rapid excoriation of the sperm head within the ooplasm ([27, 28]; Fig. 3A), this strongly suggests that SOAFs does not potentiate activation by modifying heads.
Further Characterization of Detergent-Resistant SOAF Reveals Additional Properties Distinct from Those of Cytosolic Oocyte-Activating Function
The identification of an egg-activating function that is detergent resistant (SOAF) strongly suggests that it is distinct from the cytosolic sperm factors whose prototype is glucosamine 6-phosphate isomerase-oscillin [29]. We adopted two approaches to compare SOAF and cytosolic sperm factors from a single species. First, we applied the Core Protocol to golden hamster sperm and then performed incubation at 27°C in NIM-15 mM DTT. This preparation was capable of trans-complementing heat-inactivated mouse sperm heads sufficient for activation of 100% (5 of 5) of surviving oocytes injected. A freeze-thaw protocol originally utilized in the isolation of the cytosolic, Ca2+-mobilizing activity from hamster [10, 30] liberated a hamster cytosolic activity that readily activated 100% of mouse eggs when injected alone (Table 4). This is consistent with the coexistence of SOAF and glucosamine 6-phosphate isomerase-oscillin in hamster sperm. Second, we adapted the protocol to the isolation of mouse sperm cytosolic factors. Using this method, we found that mouse sperm indeed possessed a soluble, cytoplasmic function that activated approximately 40% of oocytes when injected alone (Table 4). This level is possibly < 100% due to a combination of 1) relatively low sperm density (3 x 108 sperm/ml compared to ~109 sperm/ml for hamster; 2) the comparatively small size of mouse sperm head soluble compartments, particularly the principal segment of the acrosome [1]; and 3) the lability of the activity at 25°C—in one experiment, activity dropped from 50% (7 of 14 activated) after approximately 50 min to 10% (1 of 10 activated) after approximately 100 min. In contrast, SOAFs activity is stable at 25°C for at least several hours (data not shown). Cytosolic activity was also readily demonstrable by coinjection of the soluble fraction either with sperm fragments that had been subjected to 48°C for 30 min or with amine-modified latex beads (Table 4). In marked contrast to the freeze-thaw-generated, cytosolic fraction, injection of SOAFs in the absence of sperm heads consistently failed to activate (Table 3). This failure was exhibited even when SOAFs was isolated from the relatively high density of > 5 x 108 porcine sperm/ml and injected immediately after preparation; 0% (0 of 6) of oocytes became activated when this SOAFs was injected alone, but 100% (5 of 5) became activated when injected in the presence of inactivated mouse sperm heads.
In all cases, mouse sperm heads subjected to the freeze-thaw disruption of cytoplasmic compartments were subsequently capable of oocyte activation, even after they had been subjected to Triton X-100 extraction in the Core Protocol (Table 4). Hence, sperm heads subjected to freeze-thawing retained the ability to initiate activation via native matrix and other components. Moreover, 5 of 5 oocytes were activated by the injection of heads prepared by the Core Protocol with 5 mg/ml BSA included throughout (to minimize "stickiness" of the heads). Continued inclusion of 5 mg/ml BSA had no discernible effect on the generation of SOAFs (in NIM-15 mM DTT at 30°C), which activated 100% (9 of 9) of oocytes when injected with 48°C-treated sperm heads. Demembranated sperm heads are therefore not merely an adhesive vehicle for cytosolic, sperm-derived factors such as glucosamine 6-phosphate isomerase-oscillin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This work shows that spermatozoa contain at least two distinctive functions capable of initiating oocyte activation: a cytosolic activity and a complex submembrane activity that includes SOAF. We have demonstrated the coexistence of these functions in both the mouse and hamster. Presumably, the cytosolic function corresponds to previously described activities whose prototype is glucosamine 6-phosphate isomerase-oscillin [29, 31].
It is possible that although sperm possess two distinct functions capable of inducing activation, one of the functions in fact plays a different physiological role in fertilization. This is plausible given the involvement of an additional key Ca2+ mobilization step within sperm during the fertilization process: the induction of acrosomal exocytosis. This Ca2+-dependent process is apparently mediated via T-type, voltage-sensitive Ca2+ channels [32]. However, such channels mediate small, transient Ca2+ influxes into the acrosome, rather than the sustained, high levels observed during acrosomal exocytosis. Spermatozoa may therefore possess effectors of elevated Ca2+ levels (which work by interacting—perhaps indirectly—with inositol 1,4,5-triphosphate receptors) that, when injected into oocytes, cause Ca2+ oscillations and activation. This would have parallels with the fashion in which the nonphysiological agent SrCl2 induces Ca2+ oscillations and oocyte activation. We reason that the cytosolic activity in our study would be far more likely to be involved in acrosomal exocytosis than a relatively insoluble ensemble (which includes SOAF) derived from the nucleus/perinuclear matrix. Moreover, neither our work, nor any that we are aware of, has demonstrated cytosolic oocyte-activating factors in acrosome-reacted spermatozoa or in any sperm compartment known to traverse the oolemma following membrane fusion at fertilization.
In contrast, we have demonstrated the presence in mouse sperm of at least two molecular species that coordinately induce oocyte activation from structures known to enter the ooplasm at fertilization. We believe that the properties of SOAF described here suggest a novel mechanism of mammalian oocyte activation that we have partially reproduced in vitro, as represented in Figure 1.
In this molecular model, SOAFs is generated from SOAFm immediately after sperm-egg fusion, consistent with the rapid dispersal of perinuclear material on gamete fusion ([1, 27, 28, 33]; Fig. 3). The immediate SOAFm SOAFs transition generated on contact of the perinuclear matrix with the reducing environment of the ooplasm would rapidly liberate SOAFs, thereby initiating the first steps of activation. This SOAFm SOAFs transition is simulated in vitro by the incubation of demembranated sperm under reducing conditions (Fig. 1). It is as yet unclear whether physiologically active, native SOAF substantially exists (in some species, at least) as SOAFs in acrosome-reacted spermatozoa prior to gamete fusion, although the existence of such material would clearly favor the rapid delivery of an activating signal. Otherwise, SOAFm would presumably reside on the periphery of the perinuclear matrix so that it immediately engaged with the ooplasm at fertilization. Few, if any, molecular markers with this localization have been reported, with the possible exception of an epitope recognized by monoclonal antibody MN13 [34]. However, synchrony between mouse sperm perinuclear matrix deposition [35] and innate activating ability [16] is consistent with a perinuclear localization for SOAF. After fertilization, subsequent "waves" of SOAFs could potentially be provided through the continued solubilization of SOAFm as the perinuclear matrix became progressively excoriated by the ooplasm. In this scenario, the sperm head is a "SOAF bomb."
Our observations are perhaps best explained if the synergistic action of each molecular species (SOAFs and the heat-stable component) worked by stimulating convergent activation pathways within the oocyte. Since inactivated sperm heads prime oocytes for activation by SOAFs for several hours following their injection, any signal originating from them would either be continuous or be "memorized" by the ooplasm for that duration. The development of the trans-complementation assay described here is further enabling us to dissect ooplasmic interactions with sperm components at fertilization and with somatic chromatin following in vitro nuclear transfer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 808 956 5474.
Accepted: October 13, 1998.
Received: August 13, 1998.
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6ß1 functions as a sperm receptor. Cell 1995; 81:1095–1104.[CrossRef][Medline]This article has been cited by other articles:
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  S.-Y. Yoon and R. A Fissore Release of phospholipase C {zeta}and [Ca2+]i oscillation-inducing activity during mammalian fertilization Reproduction, November 1, 2007; 134(5): 695 - 704. [Abstract] [Full Text] [PDF]
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 N. Yoshida, M. Amanai, T. Fukui, E. Kajikawa, M. Brahmajosyula, A. Iwahori, Y. Nakano, S. Shoji, J. Diebold, H. Hessel, et al. Broad, ectopic expression of the sperm protein PLCZ1 induces parthenogenesis and ovarian tumours in mice Development, November 1, 2007; 134(21): 3941 - 3952. [Abstract] [Full Text] [PDF]
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 A. T. H. Wu, P. Sutovsky, G. Manandhar, W. Xu, M. Katayama, B. N. Day, K.-W. Park, Y.-J. Yi, Y. W. Xi, R. S. Prather, et al. PAWP, a Sperm-specific WW Domain-binding Protein, Promotes Meiotic Resumption and Pronuclear Development during Fertilization J. Biol. Chem., April 20, 2007; 282(16): 12164 - 12175. [Abstract] [Full Text] [PDF]
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 K.-B. Lee and K. Niwa Fertilization and Development In Vitro of Bovine Oocytes Following Intracytoplasmic Injection of Heat-Dried Sperm Heads Biol Reprod, January 1, 2006; 74(1): 146 - 152. [Abstract] [Full Text] [PDF]
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S. Kishigami, S. Wakayama, N. Van Thuan, and T. Wakayama Similar Time Restriction for Intracytoplasmic Sperm Injection and Round Spermatid Injection into Activated Oocytes for Efficient Offspring Production Biol Reprod, June 1, 2004; 70(6): 1863 - 1869. [Abstract] [Full Text] [PDF]
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G. Manandhar and K. Toshimori Fate of Postacrosomal Perinuclear Theca Recognized by Monoclonal Antibody MN13 after Sperm Head Microinjection and Its Role in Oocyte Activation in Mice Biol Reprod, February 1, 2003; 68(2): 655 - 663. [Abstract] [Full Text] [PDF]
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 K. Mizuno, K. Hoshi, and T. Huang Fertilization and embryo development in a mouse ICSI model using human and mouse sperm after immobilization in polyvinylpyrrolidone Hum. Reprod., September 1, 2002; 17(9): 2350 - 2355. [Abstract] [Full Text] [PDF]
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L. Keskintepe, G. Pacholczyk, A. Machnicka, K. Norris, M. A. Curuk, I. Khan, and B. G. Brackett Bovine Blastocyst Development from Oocytes Injected with Freeze-Dried Spermatozoa Biol Reprod, August 1, 2002; 67(2): 409 - 415. [Abstract] [Full Text] [PDF]
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Y.H. Choi, C.C. Love, Y.G. Chung, D.D. Varner, M.E. Westhusin, R.C. Burghardt, and K. Hinrichs Production of Nuclear Transfer Horse Embryos by Piezo-Driven Injection of Somatic Cell Nuclei and Activation with Stallion Sperm Cytosolic Extract Biol Reprod, August 1, 2002; 67(2): 561 - 567. [Abstract] [Full Text] [PDF]
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C. M. Saunders, M. G. Larman, J. Parrington, L. J. Cox, J. Royse, L. M. Blayney, K. Swann, and F. A. Lai PLC{zeta}: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development Development, August 1, 2002; 129(15): 3533 - 3544. [Abstract] [Full Text] [PDF]
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 A. C. Gordo, M. Kurokawa, H. Wu, and R. A. Fissore Modifications of the Ca2+ release mechanisms of mouse oocytes by fertilization and by sperm factor Mol. Hum. Reprod., July 1, 2002; 8(7): 619 - 629. [Abstract] [Full Text] [PDF]
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J. Cozzi, F. Monier-Gavelle, N. Lievre, M. Bomsel, and J.P. Wolf Mouse Offspring after Microinjection of Heated Spermatozoa Biol Reprod, November 1, 2001; 65(5): 1518 - 1521. [Abstract] [Full Text] [PDF]
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K. D. Campbell, W. A. Reed, and K. L. White Ability of Integrins to Mediate Fertilization, Intracellular Calcium Release, and Parthenogenetic Development in Bovine Oocytes Biol Reprod, June 1, 2000; 62(6): 1702 - 1709. [Abstract] [Full Text]
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