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Transgenic RNA Interference Reveals Role for Mouse Sperm Phospholipase C in Triggering Ca²⁺ Oscillations During Fertilization¹

Center for Research on Reproduction and Women's Health,⁴ Department of Biology,⁵ Department of Obstetrics and Gynecology,⁶ University of Pennsylvania, Philadelphia, Pennsylvania 19104 Department of Veterinary and Animal Sciences,⁷ University of Massachusetts, Amherst, Massachusetts 01003

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
A sperm-specific phospholipase (PL) C, termed PLC, is proposed to be the soluble sperm factor that induces Ca²⁺ oscillations in mammalian eggs and, thus, initiates egg activation in vivo. We report that sperm from transgenic mice expressing short hairpin RNAs targeting PLC mRNA have reduced amounts of PLC protein. Sperm derived from these transgenic mice trigger patterns of Ca²⁺ oscillations following fertilization in vitro that terminate prematurely. Consistent with the perturbation in patterns of Ca²⁺ oscillations is the finding that mating of transgenic founder males to females results in lower rates of egg activation and no transgenic offspring. These data strongly suggest that PLC is the physiological trigger of Ca²⁺ oscillations required for activation of development.

calcium, fertilization, gamete biology, ovum, sperm

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
A universal feature of fertilization in eggs of all species studied to date is a transient elevation in intracellular Ca²⁺ concentration. In mammals, sperm evoke a series of repetitive Ca²⁺ oscillations that persist for several hours and terminate with pronucleus formation [1, 2]. This pattern of repetitive Ca²⁺ oscillations in mice is essential for both early events of egg activation (e.g., resumption of meiosis, cortical granule exocytosis, recruitment of maternal mRNAs) and the developmental program [3, 4].

Fertilization likely triggers the increase in intracellular Ca²⁺ via phospholipase (PL) C-catalyzed production of inositol 1,4,5-trisphosphate (IP₃) in the egg [5]. The IP₃ then acts on type 1 IP₃ receptors to initiate Ca²⁺ release. How the fertilizing sperm activates PLC has been intensively studied for the past 15 yr. During this time, the paradigm has shifted from a sperm ligand-egg receptor model using either G protein-coupled or tyrosine kinase receptors to the proposal that a soluble, sperm-derived protein factor is responsible for activating PLC and, thereby, evoking Ca²⁺ oscillations in mammalian eggs [6]. The seminal observations leading to the new model are that fusion of the mouse sperm and egg precedes the onset of oscillations by approximately 1–5 min [7] and that injecting mouse sperm, which causes fertilization-like Ca²⁺ oscillations, supports full-term development [8].

Recent studies suggest that a novel, sperm-specific PLC, termed PLC, is the sperm factor. Expression of PLC cRNA in mouse eggs that results in synthesis of PLC estimated to be equivalent to the amount in a single sperm triggers Ca²⁺ oscillations that closely resemble the pattern observed following fertilization [9, 10]. Furthermore, immunodepleting PLC from sperm extracts abolishes the Ca²⁺-releasing activity, suggesting that PLC is the sole Ca²⁺-releasing component [9]. Finally, recent biochemical studies using recombinant PLC protein demonstrated that its activity is approximately 70% of maximal at 100 nM Ca²⁺, the resting level of Ca²⁺ in the egg [11]. This finding supports the model in which the sperm delivers a PLC that is readily activated on exposure to the egg's cytoplasm. Nevertheless, despite this evidence that PLC is the soluble sperm factor, a physiological role for PLC during egg activation in vivo has not been established. We report here that reducing sperm PLC protein by a transgenic RNA interference (RNAi) approach significantly perturbs the calcium oscillatory behavior of eggs inseminated with these sperm. This finding provides direct evidence that PLC serves as the sperm factor in vivo.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Generation of Constructs and Transgenic Mice

A polymerase chain reaction (PCR)-based strategy (PCR SHAGging) for generating RNA polymerase III (U6 small nuclear RNA promoter)-driven transgenic constructs expressing 29-base pair (bp) short hairpin RNA (shRNA) targeting PLC (nucleotides 163–191 and 1675–1703; GenBank accession no. AF435950) was employed (http://www.cshl.org/public/SCIENCE/hannon.html). The mouse U6 promoter from pSilencer 1.0-U6 small interfering RNA (siRNA) Expression Vector (Ambion, Austin, TX) was amplified using a forward primer, 5'-TCACTATAGGGCGAATTGGG-3', and one of two reverse primers containing different shRNA sequences, 5'-AAAAAAGTGATTCTTCCTTGGCTCCGTCTGCCACTCAAGCTTCAATGACAGACAGAACCAAGGAAGAATCACGGGGCCCAAACAAGGCTTTT-3' and 5'-AAAAAAGCACTATTCCTAACAACACGAATCTGCCGCAAGCTTCCAGCAGACTCGTGTTGTTAAGAATAATGCGGGGCCCAAACAAGGCTTTT-3' (bold represents shRNA sequences). The PCR was performed using puReTaq Ready-To-Go PCR Beads (Amersham Biosciences, Piscataway, NJ) in a 25-µl reaction as follows: an initial denaturation step at 95°C for 5 min, followed by 30 cycles at 95°C for 30 sec, 52°C for 30 sec, and 72°C for 20 sec, and then a final extension at 72°C for 5 min. The PCR product was cloned by TA cloning into pCR2.1 (Invitrogen, Carlsbad, CA). Constructs were excised using SacI and DraIII restriction endonucleases and gel purified using a Qiagen Gel Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Equal amounts of both constructs were mixed, and the final concentration of DNA was adjusted to 5 ng/µl. The DNA mixture was submitted to the Transgenic and Chimeric Mouse Facility at the University of Pennsylvania to generate transgenic animals in B6SJLF1/J mice.

To identify transgenic mice, PCR was performed on tail DNA. The forward and reverse primers were 5'-CGTTCTAGAACTAGTGGATCCG-3' (U6 promoter) and 5'-CCTCTAGATGCATGCTCGAGC-3' (pCR2.1 vector sequence downstream of transgenic construct), respectively, to generate an approximately 500-bp product. The PCR was performed using puReTaq Ready-To-Go PCR Beads in a 25-µl reaction as follows: an initial denaturation step at 95°C for 5 min, followed by 40 cycles at 95°C for 30 sec, 67°C for 30 sec, and 72°C for 20 sec, and then a final extension at 72°C for 5 min.

Collection of Gametes

The CF-1 female mice (age, 6–8 wk) were superovulated by sequential injections of 5 IU of eCG followed by injection of 5 IU of hCG as previously described [12]. Metaphase II eggs were collected into Hepes-buffered Whitten medium [13] containing 0.01% polyvinyl alcohol (PVA; average molecular weight, 30 000–70 000) at 13.5 h post-hCG, and cumulus cells were removed by hyaluronidase treatment. Eggs were cultured in 50-µl drops of Whitten medium containing 0.01% PVA under paraffin oil at 37°C in a humidified atmosphere of 5% CO₂ in air. Zona pellucida (ZP)-free eggs were obtained by removing the ZP with acidic Tyrode solution [14].

Sperm from nontransgenic and transgenic founder males were obtained by mating males with superovulated CF-1 females immediately following the injection of hCG. Every 0.5–1.0 h following mating, females were checked for the presence of a vaginal plug. Immediately following the detection of a plug, the female was killed, and the entire reproductive tract was removed, washed once in Hepes-buffered Whitten medium, and then transferred into either PBS or Whitten medium supplemented with 15 mg/ ml of BSA. Small incisions were made throughout the uterus to allow spermatozoa to swim out. On average, 3.5 x 10⁶ sperm were collected from each female. For in vitro fertilization (IVF), sperm were capacitated for 2 h in Whitten medium containing 15 mg/ml of BSA. All animal experiments were approved by the Institutional Animal Use and Care Committee and were consistent with National Institutes of Health guidelines.

IVF and Ca²⁺ Imaging

In all cases, IVF was performed using eggs collected from four to five superovulated CF-1 females and pooled to avoid potential bias because of inherent differences in eggs from different females. Furthermore, Ca²⁺ oscillation patterns were recorded on the same day from the same pool of eggs when comparing nontransgenic sperm to transgenic sperm; the sperm used first were alternated to control for differences caused by the age of the eggs (time after hCG administration).

The ZP-free eggs were incubated in Whitten medium containing 0.01% PVA, 10 µM fura-2-acetoxymethylester (Fura-2; Molecular Probes, Inc., Eugene, OR), and 0.025% Pluronic F-127 [15] at 37°C in an atmosphere of 5% CO₂ in humidified air for 20 min. The Fura-2-loaded eggs were transferred into a 4-µl drop of Whitten medium without BSA and placed on a temperature-controlled microscope stage under laminar flow of 5% CO₂ in air. After 10–15 min to allow the eggs to stick to the coverslip, 4 µl of Whitten medium containing 30 mg/ml of BSA were added to this drop to achieve a final BSA concentration of 15 mg/ml. For insemination, sperm were added to a final concentration of 100 000 sperm/ml. Measurements of intracellular Ca²⁺ were carried out as described previously [16], and the y axes shown in Figure 2 are the 340nm/380nm emission ratio.

View larger version (30K): [in this window] [in a new window]   FIG. 2. The Ca2+ oscillatory behavior of mouse eggs fertilized with sperm from nontransgenic (NTG; solid lines) and transgenic (TG; dashed lines) mice. Data obtained from TG 37 (A) and from TG 39 (B) are displayed. Percentages refer to the fraction of eggs exhibiting the representative patterns. The letters a, b, and c refer to eggs that oscillated for 90 min or longer, 50–89 min, and 10–49 min, respectively. The letter d refers to eggs that exhibited a single transient of normal amplitude, whereas the letter e refers to eggs that exhibited an abortive single transient. The lower part displays the data in a rank-order fashion for each egg as a function of how long it exhibited Ca2+ oscillations

Immunblot Analysis

Rabbit antiserum was raised against a 19-amino acid sequence (GYRRVPLFSKSGANLEPSS) at the C-terminus of mouse PLC [9]. The antiserum was affinity-purified using the antigenic peptide. Extracts of 0.2–1.0 x 10⁶ sperm/lane were subjected to SDS-PAGE, and the resolved proteins were transferred to a nitrocellulose membrane. The membranes were blocked with milk, and immunoblotting was performed using the anti-PLC antibody (final concentration, 0.27 µg/ml) and a horseradish peroxidase-labeled secondary antibody. AKAP4 was detected using a polyclonal rabbit anti-AKAP4 (a kind gift of Dr. Stuart Moss, University of Pennsylvania) as previously described [17] except that the primary antibody was used at a dilution of 1:1000 or 1:5000. Immunoreactivity was detected using enhanced chemiluminescence (Amersham) and x-ray film (Eastman Kodak, Rochester, NY). To determine siRNA specificity, membranes were also probed with PLC4 antiserum used at a 1:1000 dilution; the anti-PLC4 was a kind gift from Dr. Kiyoko Fukami (Tokyo University of Pharmacy and Life Science, Tokyo, Japan). For quantification of the data, the x-ray film was scanned and the file analyzed using Adobe PhotoShop (San Jose, CA). Boxes of the same size were drawn around the appropriate band, and the average pixel intensity was measured. The relative amount of each PLC isoform was calculated as the ratio of its average pixel intensity to that of the AKAP4 loading control and then expressed relative to that of the nontransgenic control.

Statistical Analysis

Statistical analysis was performed using PRISM 4.0 software (GraphPad Software, San Diego, CA). The Ca²⁺ oscillation data were analyzed using the Student t-test. Embryonic development in vitro was analyzed using the Fisher exact test and litter size using the Mann-Whitney U-test. Data are presented as mean ± SEM throughout.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
A transgenic RNAi approach exploiting an shRNA under control of the RNA polymerase III-driven U6 promoter (Fig. 1A) was used to reduce production of the sperm-specific PLC during spermatogenesis. Sperm extracts obtained from the two transgenic founder male mice (founders 37 and 39) contained approximately 60% of the amount of PLC compared to sperm extracts derived from a nontransgenic littermate (Fig. 1B). The transgenic sperm extracts were obtained from female uteri after mating and, therefore, were contaminated with other cell types. For this reason, we used the relative amount of AKAP4 in each extract to normalize for the amount of sperm-derived protein; AKAP4 is a sperm-specific protein that is a major component of the fibrous sheath [17]. Targeting of PLC appeared to be specific, because no change was apparent in the amount of PLC4, an isoform highly related to PLC and essential for the ZP-induced acrosome reaction in mice [18] (Fig. 1B). The reduction in PLC protein in individual sperm may be greater than that observed in the immunoblot, because mosaicism of the germline is common in transgenic founders. Unfortunately, the anti-PLC antibodies developed to date have been unsuitable for immunocytochemical analysis to establish the degree of mosaicism, relative decrease in PLC protein as well as the variability of this decrease between sperm, and localization of the protein.

View larger version (39K): [in this window] [in a new window]   FIG. 1. Transgenic RNAi specifically reduces PLC expression in mouse sperm. A) Schematic of PLC shRNA transgenes showing the antisense and sense sequences that were used to target PLC mRNA (seq 1 and 2). The predicted hairpin structures of shRNAs 1 and 2 are shown. B) Immunoblot analysis of sperm extracts from nontransgenic (NTG) and transgenic (TG) founders 37 and 39 for PLC and PLC4. When normalized for the amount of AKAP4 protein in the extracts, the amount of PLC in TGs 39 and 37 was 63% and 56%, respectively, of the NTG control for the shown immunoblot. The experiment was performed five times for each TG founder, and an approximately 40% reduction in the amount of PLC was consistently observed in both TG lines. The bottom two panels demonstrate that no decrease occurred in the amount of PLC4. When normalized for the amount of AKAP4 protein in the extracts, the amount of PLC4 in TGs 39 and 37 was 113% and 132%, respectively, of the NTG control for the shown immunoblot. The experiment was performed two times for each TG founder, and no reduction in the amount of PLC4 was observed in both TG lines

Sperm from transgenic founders or their littermates were collected for analysis from the reproductive tracts of mated females so that the founder males could be used repeatedly. Sperm from transgenic founders were similar in number, morphology, and motility compared to sperm from nontransgenic littermates. To determine whether a reduction in PLC results in perturbations in Ca²⁺ oscillatory behavior during fertilization, sperm from transgenic and nontransgenic males were collected and used for IVF, and the resulting Ca²⁺ oscillations were monitored. A key feature of fertilization-induced Ca²⁺ release is that the initial Ca²⁺ transient is higher in amplitude and longer in duration than the subsequent Ca²⁺ spikes. The amplitude and/or duration of the first Ca²⁺ transient in eggs fertilized with transgenic sperm differed from that observed in control fertilized eggs. With respect to founder 37, the amplitude was smaller compared to control eggs (0.58 ± 0.03 vs. 0.76 ± 0.03, respectively; P < 0.05), and 10% of these eggs (4/42) exhibited an abortive first Ca²⁺ spike of minimal amplitude, which was never observed in controls (0/58) or in founder 39 (0/25) (Fig. 2). Although no significant difference was observed in the amplitude of the first Ca²⁺ transient in eggs fertilized with sperm from founder 39 compared to controls (0.76 ± 0.02 vs. 0.84 ± 0.03, respectively; P = 0.05), the duration was shorter (5.6 ± 0.4 vs. 7.9 ± 0.2 min, respectively; P < 0.05). These results demonstrate that PLC plays an important role in shaping both the amplitude and the duration of the first Ca²⁺ transient.

The frequency and persistence of subsequent Ca²⁺ oscillations in eggs fertilized with sperm from transgenic and nontransgenic mice were determined by monitoring the inseminated eggs for 2–3 h. The relative frequency of Ca²⁺ oscillations in eggs fertilized with sperm from nontransgenic and transgenic males was not different (data not shown). The persistence, however, was markedly aberrant in eggs fertilized with sperm from both transgenic males. Eggs fertilized with sperm from transgenic founders 37 and 39 oscillated for substantially shorter periods of time compared to fertilized control eggs (40 ± 6.2 vs. 67 ± 6.0 min and 34 ± 7.0 vs. 53 ± 6.1 min, respectively; P < 0.05) (Fig. 2). Many of the eggs fertilized by sperm from transgenic males exhibited only a few Ca²⁺ rises and failed to establish persistent oscillations. These differences in the persistence of Ca²⁺ oscillations are seen clearly when these eggs are ranked with respect to the total amount of time they oscillated (Fig. 2). These data demonstrate that PLC is required to drive persistent Ca²⁺ oscillations following fertilization in vitro, and they suggest a continued role for PLC beyond initiating Ca²⁺ oscillations. Nevertheless, this effect might be indirect; for example, PLC may set later oscillation patterns by its effect on characteristics of the initial Ca²⁺ transient.

Perturbations in Ca²⁺ oscillations negatively impact egg activation and embryonic development [4, 19, 20]. Because of the aforementioned effects on Ca²⁺ oscillations using sperm from transgenic males, we determined the developmental potential of eggs fertilized with these sperm. Female mice were mated with transgenic and nontransgenic males, and fertilized eggs were collected when pronuclei started to form (19 h post-hCG). Fertilized eggs were then cultured for 5 days, and their development was monitored. A high percentage of control fertilized eggs formed pronuclei, cleaved, and developed to the blastocyst stage (81/81 [100%], 80/81 [99%], and 73/81 [90%], respectively). In contrast, many eggs fertilized after mating with founder 37 had a fertilization cone and/or a second polar body but failed to form pronuclei, resulting in a lower incidence of pronucleus formation (35/48 [73%], P < 0.05). As a consequence, fewer 1-cell embryos cleaved (35/48 [73%]), and fewer developed to the blastocyst stage (31/48 [65%], respectively). Representative images of eggs fertilized with sperm from nontransgenic and transgenic founder 37 are shown in Figure 3, A and B. Note that sperm and egg fusion proceeded normally but that egg activation did not occur (Fig. 3C). This arrested phenotype was observed in all three experiments using founder 37 (28/144 [19%]) but rarely with the control (1/191 [0.5%]). No significant difference was observed in the incidence of pronucleus formation, cleavage, and development to the blastocyst stage when founder 39 was used (114/122 [93%], 114/122 [93%], and 99/122 [81%], respectively).

View larger version (60K): [in this window] [in a new window]   FIG. 3. Phase-contrast and fluorescent images of mouse eggs fertilized in vivo. Representative images of mouse eggs fertilized with sperm from a nontransgenic (NTG) littermate (A) and transgenic (TG) founder 37 (B) are shown. All control fertilized eggs formed pronuclei, whereas eggs fertilized with sperm from TG 37 formed fewer pronuclei. Examples of pronuclei are indicated by arrows. Also shown is an egg without a pronucleus but with a fertilization cone (arrowhead) and a representative image (C) of a mouse egg fertilized in vivo with a sperm from TG 37 that failed to form pronuclei. The zygote, which was stained with 4',6'-diamidino-2-phenylindole (Tris-like), reveals the presence of a decondensed sperm head within a fertilization cone. This phenotype was consistently observed in three separate experiments. Bar = 50 µm (A and B) and 20 µm (C)

To determine whether perturbations in Ca²⁺ oscillatory behavior have an effect on postimplantation development, transgenic and nontransgenic males were mated to CF-1 females, and average litter size was evaluated. Females mated to founder 37 had litter sizes comparable to those of females mated with nontransgenic males (12 ± 1.3 vs. 13 ± 0.6, n = 6, P > 0.05). In contrast, females mated to founder 39 produced fewer offspring (7 ± 1.4 vs. 13 ± 0.6, n = 6, P < 0.05). Because transgenic founders can display a broad degree of germline mosaicism, we expected a variable subpopulation of nontransgenic sperm in our founder mice that would affect litter size. Moreover, we predicted that sperm harboring the transgene would be less likely to support development to term because of the observed perturbations in Ca²⁺ oscillatory behavior. In fact, genotypic analysis of the offspring derived from transgenic founders revealed that no transgenic offspring were born from founder 37 (0/48) and founder 39 (0/21). The absence of transgenic offspring most likely is a manifestation of a mosaic germline in both of these founders, and it strongly suggests that eggs fertilized by transgenic sperm that fail to induce normal Ca²⁺ oscillations do not develop to term, a finding that is consistent with a role for proper calcium oscillatory behavior in supporting long-term development[4].

Our results using a transgenic RNAi approach provide direct evidence that PLC is critical for triggering Ca²⁺ oscillations following fertilization, and they could have implications for human infertility and contraceptive development. Moreover, these results demonstrate the feasibility of this transgenic RNAi approach to study the function of the many male germ cell-specific genes.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Ken-ichi Sato of Kobe University (Kobe, Japan) for assisting in preparing and purifying the anti-PLC antibody and Brian Jones for technical assistance.

	FOOTNOTES

 
¹ Supported by grants from the National Institutes of Health (HD22732 to R.M.S. and C.J.W. and HD042498 to R.A.F.) and U.S. Department of Agriculture (02-2078 to R.A.F.). J.K. was supported by training grant T32 HD007305.

² Correspondence: Carmen J. Williams, Center for Research on Reproduction and Women's Health, School of Medicine, University of Pennsylvania, 1313 BRB II/III, 421 Curie Blvd., Philadelphia, Pennsylvania 19104-6080. FAX: 215 573 7627; cjwill{at}mail.med.upenn.edu

³ These authors contributed equally to this work

Received: 14 September 2004.

First decision: 18 October 2004.

Accepted: 18 November 2004.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

1. Jones KT, Carroll J, Merriman JA, Whittingham DG, Kono T. Repetitive sperm-induced Ca²⁺ transients in mouse oocytes are cell cycle dependent. Development 1995 121:3259-3266[Abstract]
2. Marangos P, Fitz-Harris G, Carroll J. Ca²⁺ oscillations at fertilization in mammals are regulated by the formation of pronuclei. Development 2003 130:1461-1472[Abstract/Free Full Text]
3. Ducibella T, Huneau D, Angelichio E, Xu Z, Schultz RM, Kopf GS, Fissore R, Madoux S, Ozil J-P. Egg to embryo transition is driven by differential responses to Ca²⁺ oscillation number. Dev Biol 2002 250:280-291[CrossRef][Medline]
4. Ozil JP, Huneau D. Activation of rabbit oocytes: the impact of the Ca²⁺ signal regime on development. Development 2001 128:917-928[Abstract]
5. Runft LL, Jaffe LA, Mehlmann LM. Egg activation at fertilization: where it all begins. Dev Biol 2002 245:237-254[CrossRef][Medline]
6. Swann K, Lai FA. A novel signaling mechanism for generating Ca²⁺ oscillations at fertilization in mammals. BioEssays 1997 19:371-378[CrossRef][Medline]
7. Lawrence Y, Whitaker M, Swann K. Sperm-egg fusion is the prelude to the initial Ca²⁺ increase at fertilization in the mouse. Development 1997 124:233-241[Abstract]
8. Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995 52:709-720[Abstract]
9. Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann K, Lai FA. PLC a sperm-specific trigger of Ca²⁺ oscillations in eggs and embryo development. Development 2002 129:3533-3544[Abstract/Free Full Text]
10. Yoda A, Oda S, Shikano T, Kouchi Z, Awaji T, Shirakawa H, Kinoshita K, Miyazaki S. Ca²⁺ oscillation-inducing phospholipase C expressed in mouse eggs is accumulated to the pronucleus during egg activation. Dev Biol 2004 268:245-257[CrossRef][Medline]
11. Kouchi Z, Fukami K, Shikano T, Oda S, Nakamura Y, Takenawa T, Miyazaki S. Recombinant phospholipase C{zeta} has high Ca²⁺ sensitivity and induces Ca²⁺ oscillations in mouse eggs. J Biol Chem 2004 279:10408-10412[Abstract/Free Full Text]
12. Kurasawa S, Schultz RM, Kopf GS. Egg-induced modifications of the zona pellucida of mouse eggs: effects of microinjected inositol 1,4,5-trisphosphate. Dev Biol 1989 133:295-304[CrossRef][Medline]
13. Whitten WK. Nutrient requirements for the culture of preimplantation mouse embryo in vitro. Adv Biosci 1971 6:129-139
14. Bornslaeger EA, Schultz RM. Adenylate cyclase activity in zona-free mouse oocytes. Exp Cell Res 1985 156:277-281[CrossRef][Medline]
15. Poenie M, Alderton J, Steinhardt R, Tsien R. Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science 1986 233:886-889[Abstract/Free Full Text]
16. Xu Z, Williams CJ, Kopf GS, Schultz RM. Maturation-associated increase in IP3 receptor type 1: role in conferring increased IP3 sensitivity and Ca²⁺ oscillatory behavior in mouse eggs. Dev Biol 2003 254:163-171[CrossRef][Medline]
17. Carrera A, Gerton GL, Moss SB. The major fibrous sheath polypeptide of mouse sperm: structural and functional similarities to the A-kinase anchoring proteins. Dev Biol 1994 165:272-284[CrossRef][Medline]
18. Fukami K, Nakao K, Inoue T, Kataoka Y, Kurokawa M, Fissore RA, Nakamura K, Katsuki M, Mikoshiba K, Yoshida N, Takenawa T. Requirement of phospholipase Cdelta4 for the zona pellucida-induced acrosome reaction. Science 2001 292:920-923[Abstract/Free Full Text]
19. Kline D, Kline JT. Repetitive calcium transients and the role of calcium exocytosis and cell cycle activation in the mouse egg. Dev Biol 1992 149:80-89[CrossRef][Medline]
20. Xu Z, Kopf GS, Schultz RM. Involvement of inositol 1,4,5-trisphosphate-mediated Ca²⁺ release in early and late events of mouse egg activation. Development 1994 120:1851-1859[Abstract]

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This Article Abstract Full Text (PDF) All Versions of this Article: 72/4/992    most recent biolreprod.104.036244v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Knott, J. G. Articles by Williams, C. J. Search for Related Content PubMed PubMed Citation Articles by Knott, J. G. Articles by Williams, C. J. Agricola Articles by Knott, J. G. Articles by Williams, C. J.