Gynogenetic Activation of Porcine Oocytes

Abstract

The possibility of fertilization without male contribution to the embryonic genome was investigated in pig oocytes. Mature oocytes were co-incubated with sperm, and in an attempt to prevent the incorporation of the sperm head into the ooplasm, the actin polymerization inhibitor cytochalasin B was added to the fertilization medium. We found that perturbing actin filament integrity did not affect the pattern of the sperm-induced Ca²⁺ signal or the process of cortical granule exocytosis, and it did not alter the percentage of activated oocytes compared to the control (oocytes fertilized in the absence of the inhibitor). However, over 20% of the cytochalasin B–treated oocytes formed only a single pronucleus after fertilization, indicating that the inhibitor blocked sperm head incorporation at least in some oocytes. In most cases, cytochalasin B also prevented the integration of the male chromosomes into the embryonic genome as determined by the absence of the SRY gene in the embryonic blastomeres or by the frequency of embryos showing green fluorescence after sperm from a GFP-transgenic boar was used for fertilization. Finally, the percentage of embryos that developed beyond the four-cell stage and the total number of nuclei in the resultant blastocysts were higher when oocytes reconstructed by nuclear transfer were activated by fertilization in the presence of cytochalasin B compared to the control group, where activation was induced by electroporation. These results suggest that fertilization in the presence of cytochalasin B can induce oocyte activation while it also prevents integration of the male genome into the embryo. This method has the potential to be used as an alternative to inducing embryonic development after nuclear transfer.

Introduction

Mammalian oocytes are ovulated with their cell cycle arrested at the metaphase stage of the second meiotic division (Alberio et al., 2001; Madgwick and Jones, 2007). They resume meiosis at fertilization when the sperm triggers repetitive elevations in their intracellular free calcium (Ca²⁺) concentration. The Ca²⁺ elevations are the result of cyclic release of Ca²⁺ from the endoplasmic reticulum and are believed to be responsible for the activation of the oocyte's developmental program (Jones, 1998). This repetitive nature is a principal aspect of the Ca²⁺ fertilization signal in mammals. It was suggested that the amplitude, duration, and frequency of the oscillations encode information that is important for proper embryo development (Vitullo and Ozil, 1992). The total duration of the signal is believed to be a major regulator of oocyte activation (Ozil et al., 2005), and different oscillation patterns result in different gene expression profiles in the developing embryo (Ozil et al., 2006). All of these data indicate the importance of proper Ca²⁺ signaling during oocyte activation and its long-term effects on embryo development.

A thorough understanding of the signaling mechanism that operates during fertilization is key for the improvement of a number of assisted reproductive technologies, and it is crucial for enhancing the efficiency of somatic cell nuclear transfer (SCNT). In the past two decades, SCNT has become a powerful tool to produce transgenic animals for a variety purposes (Niemann and Lucas-Hahn, 2012; Whyte and Prather, 2011). During the process of nuclear transfer, the chromosomes of a mature oocyte are replaced by those of a donor cell, and the reconstructed oocyte is then activated to induce embryo development.

Conventional methods to activate the oocyte's developmental program include either electroporation or treatment with chemicals to generate transient elevations in the oocyte's intracellular free Ca²⁺ concentration (Alberio et al., 2001; Cervera et al., 2010; Machaty, 2006). In livestock species, including swine, such treatments generally induce a single Ca²⁺ transient in the cytoplasm. Although a single Ca²⁺ transient is sufficient to activate oocytes (Machaty et al., 1997a,b), it is generally accepted that the repetitive Ca²⁺ elevations observed during fertilization offer physiological benefits over single, static Ca²⁺ rises and trigger oocyte activation more effectively (Ducibella et al., 2002; Toth et al., 2006). Numerous studies, therefore, have been conducted to improve the efficiency of SCNT by developing novel parthenogenetic activation methods capable of mimicking the signal induced by the fertilizing sperm. Despite great efforts, the activation methods currently available can generate only a single Ca²⁺ transient, which is enough to stimulate blastocyst formation and, in some cases, term development, but may contribute to the low rates of development to term. In fact, improper activation of the reconstructed oocyte may be accountable, at least in part, for the low efficiency of the nuclear transfer technology in swine (Machaty et al., 1999).

Stimulating the signal transduction pathway that is normally used by the fertilizing sperm to activate the oocyte seems to be an attractive way to induce embryo development with high efficiency. Under physiological conditions, the sperm triggers oocyte activation by introducing phospholipase Cζ (PLCζ) into the oocyte (Saunders et al., 2002). PLCζ generates periodic cycles of inositol 1,4,5-trisphosphate (InsP₃) in the ooplasm that binds to its receptors on the endoplasmic reticulum and stimulates the release of Ca²⁺ from cellular stores, thereby causing the prolonged series of Ca²⁺ oscillations (Swann et al., 2006). Artificial introduction of PLCζ into oocytes also leads to activation: When complementary RNA (cRNA) of PLCζ or the recombinant protein was microinjected into oocytes of various species, it stimulated repetitive Ca²⁺ transients and development to the blastocyst stage (Cox et al., 2002; Rogers et al., 2004; Ross et al., 2008; Yoneda et al., 2006; Yu et al., 2008). However, in the absence of commercially available PLCζ, the method currently used has limited practical application.

In the present study, we explored the possibility of inducing gynogenetic development in porcine oocytes. We reasoned that co-incubation of oocytes with spermatozoa normally leads to gamete fusion followed by the onset of the long-lasting Ca²⁺ oscillations in the oocyte. If subsequent incorporation of the sperm into the ooplasm could be prevented, the method would deliver a physiological activating signal and stimulate embryo development effectively without allowing male genetic contribution to the embryonic genome. Because cytochalasin B was previously shown to inhibit sperm incorporation into oocytes of various species (Byrd and Perry, 1980; Gould-Somero et al., 1977; Sutovsky et al., 1996), we used this actin polymerization inhibitor in combination with in vitro fertilization in our experiments. As demonstrated below, the method was efficient in triggering gynogenetic development, and it may be a viable option for activation of oocytes reconstructed during SCNT.

Materials and Methods

Chemicals

Unless otherwise stated, all chemicals and reagents used in the study were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA).

In vitro maturation of oocytes

Prepubertal gilt ovaries were obtained from a local abattoir. The cumulus–oocyte complexes (COCs) were aspirated from medium-sized follicles (3–6 mm in diameter) using a 20-gauge hypodermic needle attached to a 12-cc disposable syringe. The follicular fluid containing the COCs was placed into search dishes with HEPES-buffered Tyrode's Lactate (TL-HEPES) medium (Hagen et al., 1991), and those with several layers of cumulus cells and evenly dark cytoplasm were selected for maturation. These COCs were washed and transferred into 500 μL of Tissue Culture Medium-199 (TCM-199) supplemented with 0.1 mg/mL cysteine, 10 ng/mL epidermal growth factor (EGF), 10 IU/mL luteinizing hormone (LH), and 10 IU/mL follicle-stimulating hormone (FSH), and incubated at 39°C in 5% CO₂ in air for 44 h. After this maturation period, the COCs were collected and the cumulus cells were removed by vortexing for 5 min in TL-HEPES with 2 mg/mL hyaluronidase. Oocytes with an extruded first polar body, evenly dark cytoplasm, and an intact plasma membrane were used in the experiments.

In vitro fertilization and embryo culture

Groups of 20–30 oocytes were placed into 50-μL droplets of a modified Tris-buffered fertilization medium consisting of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl₂×2H₂O, 20 mM Tris (crystallized free base), 11 mM glucose, 5 mM sodium pyruvate, 0.1% bovine serum albumin (BSA), and 1 mM caffeine covered with mineral oil. To investigate the effect of the disruption of actin filaments on fertilization and subsequent embryo development, the fertilization medium in some cases was supplemented with 7.5 μg/mL cytochalasin B, an inhibitor of actin polymerization. Fresh semen [either from a large white boar or a transgenic pig expressing the green fluorescent protein (GFP)] was washed twice by centrifugation at 900×g for 4 min in Dulbecco's phosphate-buffered saline (DPBS). The final sperm pellet was resuspended with the fertilization. The sperm suspension was added to each 50-μL droplet containing the oocytes at a final concentration of 5×10⁵ cells/mL, and the gametes were co-incubated for 5 h. For the evaluation of embryo development, the presumptive zygotes were transferred into 20-μL droplets (10 zygotes per droplet) of PZM-3 medium (Yoshioka et al., 2002) and cultured for 6 days. The nuclei of the cultured embryos were then stained with Hoechst 33342, and the developmental stages of the embryos together with the total nuclear numbers were determined.

Measurement of intracellular free Ca²⁺ levels

The zona pellucida was removed using 0.05% protease, and the zona-free oocytes were loaded with the Ca²⁺ indicator dye Fura-2 by incubation in the presence of 2 μM Fura-2 AM and 0.02% Pluronic F-127 (both from Invitrogen) for 40–50 min. The oocytes were preincubated with porcine spermatozoa for 1 h at 39°C and transferred into a measuring chamber with a glass bottom. The chamber was then placed on the heated stage of an inverted microscope, and changes in the intracellular free Ca²⁺ concentration were recorded using InCyt Im2, a dual-wavelength fluorescence imaging system (Intracellular Imaging, Inc., Cincinnati, OH, USA). Fura-2 in the cytoplasm was excited at 340 and 380 nm using a xenon arc lamp, and the emitted fluorescence was detected at 510 nm by a CCD camera. The measurements were repeated at least 10 times for each treatment using different oocytes. The results are presented as the ratio of the 340- and 380-nm fluorescence values; ratios of 1 and 5 represent ∼100 and ∼1200 nM Ca²⁺, respectively (Lee et al., 2012).

Evaluation of cortical reaction in oocytes

Five hours after the beginning of in vitro fertilization, the zona pellucida of the potential zygotes was removed by a brief incubation in 0.05% protease. After being washed in PZM-3 medium several times, the zygotes were fixed with 4.0% paraformaldehyde in PBS for 30 min at room temperature. Nonspecific binding sites were blocked by incubation in PBS containing 3 mg/mL BSA and 100 mM glycine for 15 min. The cells were then permeabilized in PBS containing 0.1% Triton X-100 for 5 min and washed in PBS containing 3 mg/mL BSA. The cortical granules were stained with 100 μg/mL fluorescein isothiocyanate (FITC)-labeled peanut agglutinin in PBS for 30 min (Machaty et al., 2000). The zygotes were mounted on microscope slides under coverslips, and the presence or absence of cortical granules was evaluated using a Zeiss LSM 710 laser-scanning confocal microscope (Carl Zeiss MicroImaging, LLC, Thornwood, NY, USA).

Determining chromatin configuration

To evaluate whether oocyte activation had occurred and pronuclei had formed following gamete interaction, the chromatin was stained with Hoechst 33342. Sixteen hours after the beginning of in vitro fertilization, the zona pellucida was removed enzymatically from the presumptive zygotes. These zona-free zygotes were then stained with the fluorescent dye for 15 min at room temperature, mounted on microscope slides, and examined using a Nikon Eclipse 50i microscope with an epifluorescence attachment. The chromatin configuration in each cell was then established and recorded.

PCR screening of embryos

Individual blastomeres were isolated from four-cell-stage embryos produced by fertilization in the presence of cytochalasin B. For this purpose, the zona pellucida was removed enzymatically from each embryo, and the blastomeres were separated mechanically by pipetting in Ca²⁺-free TL-HEPES medium. The blastomeres were then stained with Hoechst 33342, transferred individually into 10-μL droplets of TL-HEPES, and observed briefly under ultraviolet (UV) light to confirm that they were true blastomeres containing nuclei. Those without nuclear material were considered the result of oocyte fragmentation and discarded; blastomeres with nuclei were transferred into individual PCR tubes containing lysis buffer. The tubes were incubated at 65°C for 30 min and then at 95°C for 10 min to prepare the DNA for the PCR reactions. The presence or absence of the SRY (sex-determining region Y) gene was then determined in the samples using previously published oligonucleotide primers (GenBank ID U49860.2). The primers were designed to amplify a 163-bp fragment of the porcine SRY gene (Pomp et al., 1995). They included the forward primer 5′-TGAACGCTTTCATTGTGTGGTC-3′ and the reverse primer 5′-GCCAGTAGTCTCTGTGCCTCCT-3′. For the amplification, the HotStarTaq Master Mix (Qiagen, Valencia, CA, USA) was used. The reaction started with one cycle of 95°C for 15 min followed by 39 cycles of denaturation for 45 sec at 94°C, annealing for 30 sec at 55°C, and extension for 45 sec at 72°C with a 10-min extension following the final cycle. The PCR products were separated by electropohoresis using a 2.5% agarose gel.

Somatic cell nuclear transfer

The polar body along with a portion of the adjacent cytoplasm containing the metaphase chromosomes were removed from mature oocytes and a donor cell (a fibroblast cell isolated from a day-35 porcine fetus) was placed in their perivitelline space as described earlier (Lai and Prather, 2003). The overlying plasma membranes of the recipient oocytes and the donor cells were then fused in a Ca²⁺-free fusion medium (300 mM mannitol, 0.2 mM MgSO₄, 0.5 mM HEPES, 0.1% polyvinyl alcohol) by two DC pulses (1-sec interval) at 1.2 kV/cm for 30 μsec (using a BTX Electro Cell Manipulator, Harvard Apparatus, Holliston, MA, USA). Following fusion, the reconstructed oocytes were divided into two groups. Control oocytes were activated by an electrical stimulus in an activation medium (300 mM mannitol, 1.0 mM CaCl₂, 0.1 mM MgCl₂, and 0.5 mM HEPES, pH 7.3); oocytes in the other group were activated by co-incubation with porcine spermatozoa in the presence of 7.5 μg/mL cytochalasin B for 5 h as described for in vitro fertilization. The oocytes were then washed and cultured in PZM-3 medium for 6 days.

Statistical analysis

Percentage data were compared by the chi-squared test and Ca²⁺ measurement data and total cell numbers were analyzed using Student's t-test. In all comparisons, differences with p<0.05 were considered statistically significant.

Results

Actin disruption does not prevent early events of fertilization

To explore whether early oocyte activation events at fertilization are affected by the presence of the actin filament disruptor in the fertilization medium, we monitored changes in the intracellular free Ca²⁺ concentration of the oocytes. Control oocytes (8 out of 10 measured) that were not exposed to the inhibitor showed the expected long-lasting Ca²⁺ oscillations in response to sperm (Fig. 1). The oscillations started approximately an hour after the beginning of gamete co-incubation; they occurred every 15–17 min and lasted for several hours. The sperm-induced Ca²⁺ signal had a similar pattern in oocytes that were fertilized in the presence of cytochalasin B. The frequency, amplitude, and duration of the Ca²⁺ transients were similar to those detected in the control group.

FIG. 1.

Ca²⁺ signals at fertilization. (A) Repetitive Ca²⁺ transients induced by the fertilizing sperm in a control oocyte. (B) Ca²⁺ transients in an oocyte fertilized in the presence of cytochalasin B. The sperm-induced Ca²⁺ signal was similar in these oocytes despite the presence of the inhibitor.

Next, staining with FITC-labeled peanut agglutinin was used to determine whether the Ca²⁺ signals were able to induce another event of oocyte activation, the exocytosis of the cortical granules in the cytochalasin B–treated oocytes. Most of the nonfertilized control oocytes (27/34) displayed a bright fluorescent ring below their plasma membranes, indicating the presence of cortical granules. In contrast, the majority of the fertilized oocytes, either with or without cytochalasin B treatment, lacked staining by the agglutinin, suggesting that the exocytosis of the cortical granules had completed in these cells (Table 1). Representative images of the stained oocytes are shown in Figure 2. Finally, the generation of the fertilization Ca²⁺ signal and the subsequent cortical reaction indicate that membrane fusion between the gametes had also taken place despite the action of cytochalasin B. Taken together, these data indicate that the presence of the actin polymerization inhibitor does not interfere with the induction of sperm-induced oocyte activation.

FIG. 2.

Cortical granules visualized by staining with FITC-labeled peanut agglutinin. (A) Unfertilized oocyte; (B) fertilized control oocyte; (C) oocyte fertilized in the presence of cytochalasin B. Each image represents five optical layers of the respective oocyte.

Table 1.

Cortical Granules in Porcine Oocytes As Indicated by Staining with Fluorescein Isothiocyanate–Labeled Peanut Agglutinin

Treatment	No. of oocytes	No. (%) of oocytes with cortical granules
Untreated control	34	27 (79.4)^a
Fertilization	31	9 (29.0)^b
Fertilization with cytochalasin B	33	10 (30.3)^b

a,b

Different superscripts indicate statistical differences (p<0.01).

Disruption of actin filaments inhibits sperm head incorporation in the ooplasm

We then investigated whether sperm head incorporation into the oocyte cytoplasm was altered by inhibition of actin polymerization. For this purpose, the experimental oocytes were fertilized in the presence of cytochalasin B, whereas control oocytes were fertilized under normal IVF conditions. Sixteen hours after insemination, the zona pellucida of each oocyte was removed and the chromatin stained with Hoechst 33342. We found that the percentage of activated oocytes as determined by the presence of pronuclei in the ooplasm was similar between the two groups. However, over 20% of the cytochalasin B–treated oocytes had only one pronucleus 16 h after insemination, whereas none of the control oocytes displayed only a single pronucleus at this time (p<0.001), and a significantly larger proportion of them had multiple pronuclei (p=0.005; Table 2). This shows that at least in some of the oocytes cytochalasin B prevented the incorporation of the sperm head into the ooplasm, but oocyte activation did occur following fusion of the gametes.

Table 2.

Pronuclear Formation in Oocytes after In Vitro Fertilization in the Presence or Absence of Cytochalasin B

Treatment	No. of oocytes	No. (%) of oocytes activated	No. (%) of oocytes with 1 PN	No. (%) of oocytes with >1 PN
Without cytochalasin B	84	56 (66.7)	0 (0)^a	56 (66.7)^a
With cytochalasin B	98	69 (70.4)	24 (24.5)^b	45 (45.9)^b

a,b

Different superscripts in the same column indicate differences (p<0.05).

PN, pronucleus.

Cytochalasin inhibited the integration of the male chromatin into the embryonic genome

To determine whether the embryos produced in the presence of the actin inhibitor were the result of true fertilization or were potential gynogenotes whose development was a result of the sperm-induced Ca²⁺ signal without genetic contribution from the sperm, polymerase chain reaction (PCR) analysis was conducted. Individual blastomeres were isolated from four-cell embryos, and the presence or absence of the SRY gene in the blastomeres was determined using appropriate primers. The percentage of the blastomeres that contain the SRY gene in each group is indicative of the embryos' origin. The PCR analysis revealed that in the control group the SRY gene was present in 54% of the blastomeres, whereas only 19% of the blastomeres were SRY positive in the cytochalasin B–treated group (Fig. 3; Table 3).

FIG. 3.

Amplification of a fragment of the SRY gene in individual blastomeres of embryos produced in the presence or absence of cytochalasin B. Lanes 1–7, blastomeres of embryos produced in the presence of cytochalasin B; lane 8, molecular weight marker; lanes 9–14, blastomeres isolated from control embryos. A 163-bp band indicates the presence of an amplimer of the SRY gene.

Table 3.

Results of PCR Analysis Showing the Presence of the SRY Gene in Blastomeres of Four-Cell Stage Embryos Produced by Fertilization in the Presence or Absence of Cytochalasin B

Treatment	Number of embryos	No. of blastomeres examined	No. (%) of blastomeres expressing SRY
Without cytochalasin B	30	112	61 (54.5)^a
With cytochalasin B	30	110	21 (19.1)^b

a,b

Different superscripts in the same column indicate statistical differences (p<0.01).

To confirm these findings and to further examine whether the disruption of actin filaments can prevent incorporation of the male genome into the embryo, sperm from a transgenic boar heterozygous for GFP was used for in vitro fertilization. The animal carried the GFP transgene in half of its spermatozoa. After 6 days of culture, the embryos were collected and examined under UV light. We found that 35.4% of the control embryos showed green fluorescence when exposed to UV light; this frequency was 1.9% in the group that was produced in the presence of cytochalasin B (Fig. 4; Table 4). This shows that in most cases cytochalasin B prevented sperm contribution to the embryonic genome. The data also indicate that cleavage frequency and the percentage of embryos forming blastocysts were not affected by the treatment with the inhibitor.

FIG. 4.

Images showing embryos produced using GFP-transgenic sperm. (A) Bright-field image showing one expanded blastocyst and one four-cell embryo. (B) Fluorescence image indicating that both embryos were positive for GFP. The percentage of GFP-positive embryos was significantly higher in the control group generated in the absence of cytochalasin B.

Table 4.

Frequency of GFP-Positive Embryos after Fertilization in the Presence or Absence of Cytochalasin B

Treatment	No. of embryos developing	No. (%) of GFP-positive embryos	No. (%) of blastocysts	No. (%) of GFP-positive blastocysts
Without cytochalasin B	490	146 (35.4)^a	57 (11.6)^a	35 (7.1)^a
With cytochalasin B	464	7 (1.9)^b	58 (12.5)^a	1 (0.2)^b

a,b

Different superscripts in the same column indicate statistical differences (p<0.01).

Fertilization in the presence of cytochalasin B can activate oocytes after nuclear transfer

Finally, we wanted to know whether insemination in the presence of cytochalasin B could be used to activate the developmental program of oocytes reconstructed by nuclear transfer. The percentage of cleaved oocytes in the group where activation was stimulated with sperm in the presence of cytochalasin was 72.0% and a similar cleavage frequency was detected in the control group as well (71.1%; Table 5). However, following activation with sperm plus cytochalasin B, 40.6% of the embryos developed beyond the four-cell stage, and this frequency was significantly higher than that in the electroporation group (23.2%; p<0.01%). In addition, although a similar percentage of embryos formed blastocysts in the experimental and control groups (6.3% and 8.8%, respectively), the average cell number per blastocyst was significantly higher when activation was induced with sperm plus cytochalasin B compared to the control (29.0 vs. 20.1; p<0.01).

Table 5.

Development of Embryos Produced by Nuclear Transfer Using Different Oocyte Activation Methods

Type of oocyte activation	Total no. of oocytes	No. (%) of cleaved oocytes	No. (%) of embryos beyond the four-cell stage	No. (%) of blastocysts	Mean no. of nuclei per blastocyst
Electroporation	194	138 (71.1)^a	45 (23.2)^a	17 (8.8)^a	20.1±1.2^b
IVF plus cytochalasin B	207	149 (72.0)^a	84 (40.6)^b	13 (6.3)^a	29.0±2.1^a

a,b

Different superscripts in the same column indicate differences (p<0.01).

IVF, in vitro fertilization.

Discussion

Fertilization in mammals involves a series of finely coordinated steps that eventually lead to the union of the male and female genomes and the initiation of embryonic development. It begins with the binding of the sperm to the oocyte's zona pellucida. Binding induces the acrosome reaction, which facilitates the penetration of the zona by the acrosome-reacted sperm. The male gamete then crosses the perivitelline space, and its plasma membrane at the equatorial region fuses with the oocyte plasma membrane, creating cytoplasmic continuity between the two gametes (Yanagimachi, 1994). This is followed by the incorporation of the entire sperm into the oocyte's cytoplasm. The sperm nucleus then decondenses into the male pronucleus while the oocyte's chromosomes create the female pronucleus following emission of the second polar body. The sperm aster formed by the assembly of maternal centrosomal components around the sperm centriolar complex then coordinates the union of the male and female pronuclei (Schatten, 1994).

Incorporation of the sperm into the oocyte's cytoplasm requires the motile force of the sperm tail as well as the action of microfilaments in the oocyte's cortex (Le Guen et al., 1989). This was first demonstrated in studies using oocytes of marine invertebrates such as Urechis caupo, Spisula, and sea urchin, where disruption of actin filaments with cytochalasin B prevented the incorporation of sperm into the oocyte cytoplasm (Byrd and Perry, 1980; Gould-Somero et al., 1977; Longo, 1978). In bovine oocytes, complete incorporation of the fertilizing sperm also depended on the integrity of microfilaments; disrupting the microfilaments with cytochalasin B inhibited or impaired sperm incorporation in most oocytes (Sutovsky et al., 1996). In that study, 16 h after the beginning of insemination (which under normal conditions is the time of pronuclear apposition) in most oocytes (∼73%), the sperm was still attached to the oolemma. In the rest of the cases, the sperm was incorporated into the ooplasm, but male pronuclear formation was mostly retarded, and only in 8.1% of the oocytes was the sperm head transformed into a male pronucleus. Nevertheless, activation took place in ∼95% of the oocytes, indicating that although microfilament disruption interfered with sperm incorporation and formation of the male pronucleus, gamete fusion and other downstream events of oocyte activation were not affected.

Actin is also present in the sperm. Prior to capacitation, the monomeric form (G-actin) seems to be predominant (Ochs and Wolf, 1985; Virtanen et al., 1984), and it polymerizes into F-actin during capacitation and acrosome reaction. The process is necessary for the translocation of specific glycolipids and proteins to the equatorial region that takes place at this time (Castellani-Ceresa et al., 1992; Moreno-Fierros et al., 1992; Saxena et al., 1986). Its importance is demonstrated by the fact that the disruption of actin in boar spermatozoa had a negative effect on fertilization (Castellani-Ceresa et al., 1993). In a similar manner, actin inhibition in guinea pig or human spermatozoa prevented incorporation deep into the ooplasm of zona-free hamster oocytes (Rogers et al., 1989; Sánchez-Gutiérez et al., 2002). These data indicate that actin is essential for normal fertilization, and its major role is to facilitate the incorporation of the sperm head into the ooplasm after fusion of the gametes.

The fact that intact actin filaments are needed for sperm incorporation but not for other fertilization events prompted us to hypothesize that actin disruption followed by fertilization might be an effective tool for artificial activation of oocytes. Cytochalasins B or D are used most commonly for the disruption of actin filaments. Cytochalasins are fungal metabolites that bind actin and trigger various effects in cells (Wodnicka et al., 1992). Like capping proteins, they bind to the rapidly polymerizing barbed end of the actin filaments (Brenner and Korn, 1979). It has been hypothesized that cytochalasin binding inhibits filament growth at this end, while it results in depolymerization from monomer loss at the other (pointed) end (Tanenbaum, 1978). In addition, cytochalasins were shown to cleave actin filaments and to stabilize oligomers (Gottlieb et al., 1993; Stevenson and Begg, 1994). Thus, incubation in the presence of the drug causes disruption of actin filaments in the cells. In our experiments, actin disruption did not affect the signaling cascade that operates during fertilization. The sperm-induced Ca²⁺ oscillations generated in the oocytes following a treatment with cytochalasin were similar to those observed in the control oocytes. Under physiological conditions, activation of mammalian oocytes is triggered by PLCζ, which diffuses from the sperm head into the oocyte (for a recent review, see Swann and Lai, 2013). The fact that a similar percentage of oocytes displayed the fertilization Ca²⁺ signal in both groups also indicates that membrane fusion was not altered by the presence of the inhibitor.

In addition, we evaluated whether cortical granule exocytosis, an activation event downstream of the sperm-induced Ca²⁺ signal, was influenced by the disruption of actin filaments. Cortical granules are membrane-bound organelles in the oocyte cortex. They release their contents into the perivitelline space during fertilization; the contents are thought to modify the zona pellucida and prevent polyspermy. The granules are synthesized continuously at the Golgi complexes in growing oocytes and migrate to the cortex until the time of ovulation (Cran and Cheng, 1985; Ducibella et al., 1994). Although migration depends on the presence of intact microfilaments (Kim et al., 1996) and the compensatory endocytosis that retrieves membranes deposited at the oolemma during the cortical reaction also requires actin filaments (Sokac et al., 2003), exocytosis itself (vesicle docking and membrane fusion) is mediated by N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins rather than actin (Tsai et al., 2011). As expected, actin disruption in our experiments did not interfere with cortical granule exocytosis. These data supported our hypothesis that fertilization in the presence of an actin polymerization inhibitor has the potential to induce oocyte activation.

Actin disruption at fertilization interfered with sperm head incorporation into the ooplasm: Over 20% of the cytochalasin-treated oocytes had only one pronucleus 16 h following insemination. This implies that fusion and activation occurred in these oocytes, but the male pronucleus did not form. Furthermore, previous data indicate that parthenogenetically activated pig oocytes may form more than one pronucleus (Machaty et al., 1996; Procházka et al., 1992; Suzuki et al., 2002), thus the percentage of oocytes that initiated sperm-induced gynogenetic development was probably higher than 20%. In contrast, none of the control oocytes fertilized in the absence of the inhibitor had only one pronucleus. The lack of sperm head incorporation in the ooplasm was confirmed by PCR analysis. When designing this experiment, we reasoned that the percentage of the blastomeres carrying the SRY gene would indicate the origin of the embryos. Under normal conditions, about half of the in vitro fertilization (IVF) embryos are male. Therefore, if the embryos derive from fertilization, ∼50% of the isolated blastomeres should be positive for the SRY gene. This was, in fact, the case in the control group. However, if development is stimulated by sperm fusion without sperm head incorporation, the resulting embryos carry the un-incorporated male genome in only one of their blastomeres (in four-cell embryos this is 25% of the blastomeres). Because only in half of the cases is development stimulated by a Y-bearing sperm (in the other half an X-bearing sperm fuses with the oocyte), only one out of eight isolated blastomeres (12.5%) is expected to carry the SRY gene. PCR data indicated that 19% of the blastomeres were SRY positive; this frequency is close to that expected based on previous calculations. The fact that it is slightly higher indicates that, despite the presence of the inhibitor sperm incorporation was not prevented in some oocytes and these oocytes integrated the male chromosomes into the embryonic genome.

Integration of the male genome in the embryos was further tested by using semen from a transgenic boar for in vitro fertilization. Half of the sperm collected from the animal carries the GFP transgene; therefore, ∼50% of the embryos generated by normal in vitro fertilization would be expected to show green fluorescence when exposed to UV light. At the same time, if incorporation of the male genome is successfully prevented by cytochalasin B, the resulting embryo should display no fluorescence. Only 1.9% of the embryos that had developed from the cytochalasin-treated oocytes showed green fluorescence, indicating that male contribution to the embryonic genome in these embryos was minimal, probably due to the action of the inhibitor. When evaluating the potential of this method to activate oocytes reconstructed by nuclear transfer, we obtained promising results. Cleavage frequency and blastocyst formation in the cytochalasin/sperm–treated oocytes were similar to the control where development was stimulated by electroporation. However, following insemination in the presence of cytochalasin B, a significantly higher percentage of the resulting embryos developed beyond the four-cell stage, and those that formed blastocysts had significantly more cells compared to the control. These data indicate that the method effectively stimulated oocyte activation after nuclear transfer and supported development of the cloned embryos to the blastocyst stage.

Overall, we demonstrated that fertilization in the presence of an actin polymerization inhibitor triggers oocyte activation very effectively. Cytochalasin B allows gamete fusion and the generation of the long-lasting fertilization Ca²⁺ signal that induces meiotic resumption in the oocyte. However, at least in a certain percentage of the oocytes, it causes gynogenetic development by preventing incorporation of the sperm head into the ooplasm and the integration of the male chromosomes into the genome of the developing embryo. Sperm head incorporation may not be blocked in all oocytes by the inhibitor, and some of the resulting embryos might be derived from regular fertilization. Nevertheless, on the basis of the data obtained, the method has great potential for oocyte activation in nuclear transfer programs.

Footnotes

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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Gynogenetic Activation of Porcine Oocytes

Abstract

Abstract

Introduction

Materials and Methods

Chemicals

In vitro maturation of oocytes

In vitro fertilization and embryo culture

Measurement of intracellular free Ca2+ levels

Evaluation of cortical reaction in oocytes

Determining chromatin configuration

PCR screening of embryos

Somatic cell nuclear transfer

Statistical analysis

Results

Actin disruption does not prevent early events of fertilization

Disruption of actin filaments inhibits sperm head incorporation in the ooplasm

Cytochalasin inhibited the integration of the male chromatin into the embryonic genome

Fertilization in the presence of cytochalasin B can activate oocytes after nuclear transfer

Discussion

Footnotes

Author Disclosure Statement

References

Measurement of intracellular free Ca²⁺ levels