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Biology of Reproduction 66, 1095-1103 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Porcine Sperm Factor Supports Activation and Development of Bovine Nuclear Transfer Embryos1

Jason G. Knotta,b, Kasinathan Poothapillaia,b, Hua Wua, Chang Li Hea, Rafael A. Fissorea, and James M. Robl2,,c

a Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003 b Hematech LLC, Manhattan, Kansas 66502 c Hematech LLC, Worcester, Massachusetts 01605


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
A study was undertaken to determine whether injection of porcine sperm factors (pSF), which trigger oscillations in intracellular calcium concentration ([Ca2+]i) in mammalian oocytes, could be used to activate bovine oocytes during nuclear transfer. To date, only combined treatments that induce a monotonic rise in [Ca2+]i and inhibit either phosphorylation or protein synthesis have been utilized in nuclear transfer. Several doses of pSF were tested. Injection of 5 mg/ml pSF triggered [Ca2+]i oscillations that resembled those associated with fertilization with respect to amplitude and periodicity, and as a result, a high percentage of oocytes underwent activation. Furthermore, this concentration of pSF supported in vitro and in vivo development up to 60–90 days of gestation, comparable to development in control nuclear transfer embryos. Nevertheless, neither activation procedure supported development as well as did fertilization. The effectiveness of pSF as an activating agent in bovine oocytes may have been compromised because pSF was unable to support oscillations past 3–5 h postinjection and a second injection was necessary to extend the [Ca2+]i oscillations. Likewise, a single injection of pSF failed to trigger downregulation of the inositol 1,4,5-trisphosphate receptor 1 subtype, whereas a second injection downregulated the receptor in a manner similar to that seen in fertilized oocytes. These results demonstrate that soluble factor(s) from porcine sperm can support early development in bovine nuclear transfer embryos; however, the efficacy may be limited because of the premature cessation of the induced oscillations.

assisted reproductive technology, calcium, developmental biology, embryo, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
In the last 4 years, significant progress has been achieved in cloning mammals by nuclear transfer of somatic cells. Sheep, cattle, mice, goats, and most recently pigs have been cloned from both adult and fetal cell types [1–5]. Despite all of these efforts, the efficiency of the technology remains low (reviewed in [6]). The protocol that is currently used to generate cloned cattle involves either the transfer of a G1 [2, 7] or presumably a G0 nucleus [1] into an enucleated metaphase II oocyte (MII). Following artificial activation and embryo culture in vitro, adequate numbers of transferable blastocysts can be obtained, although a significant proportion of cloned fetuses are lost between 30 and 90 days of gestation following embryo transfer in comparison to in vitro fertilization (IVF) embryos that are cultured under similar conditions [8–12]. In addition, a number of pregnancies are lost to abortion during the latter stages of gestation; abnormal placentation contributes to both early and late-term abortions [8, 12, 13]. Other abnormalities such as enlarged umbilical cords [8, 14] and oversized fetuses [2, 14] have been reported, and even fetuses that develop to term frequently experience respiratory distress and/or poor adaptation to extrauterine life [2] and may die shortly after birth [15].

One factor that may affect the development of nuclear transfer embryos is oocyte activation. When cloning mammals from somatic cells, the diploid somatic nucleus replaces both the oocyte and sperm genetic contribution; consequently, activation must be initiated artificially. Most parthenogenetic activation protocols rely on combined treatments that induce a monotonic rise in intracellular calcium concentration ([Ca2+]i) and inhibit either protein phosphorylation or protein synthesis (e.g., ethanol or calcium ionophores such as ionomycin and 6-dimethylaminopurine [6-DMAP] or cycloheximide) [16, 17]. However, at fertilization the sperm activates the oocyte by generating a series of rises in [Ca2+]i that persists for several hours [18] (reviewed in [19]). The interval between [Ca2+]i rises in mammalian oocytes is species specific, and in bovine oocytes, [Ca2+]i rises are observed on average every 20 min [20, 21]. These [Ca2+]i oscillations are responsible for cortical granule exocytosis, resumption of meiosis, and initiation of development [22] (reviewed in [23]).

Besides their role in activation, [Ca2+]i oscillations may have a beneficial impact on development. For example, simulation of multiple [Ca2+]i rises in mouse, rabbit, and cow oocytes by the application of repetitive electrical pulses resulted in rapid and persistent downregulation of maturation promoting factor activity [24], accelerated pronucleus formation [25], and increased rates of development to the blastocyst stage when compared with oocytes activated by single or only a few [Ca2+]i rises [26, 27]. Likewise, when these embryos were transferred into recipient animals, increased rates of implantation were observed [26, 28]. This improvement in developmental rates could reflect the selective expression of a number of genes whose expression may be facilitated by [Ca2+]i oscillations. Notably, in various somatic cell types, [Ca2+]i oscillations can regulate gene expression, and both the amplitude and frequency of the [Ca2+]i rises may selectively influence the expression of certain genes [29, 30]. Collectively, these studies demonstrate that [Ca2+]i oscillations may be beneficial for embryonic development and important for oocyte activation.

The sperm may induce [Ca2+]i oscillations by delivering a soluble factor that is released during the fusion of sperm and oocyte membranes [31, 32]. In support of this hypothesis, sperm and oocyte fusion precedes the induction of [Ca2+]i oscillations during fertilization [33]. Also, direct injection of sperm [34] or extracts from a variety of nonmammalian and mammalian sperm into mammalian oocytes, circumventing membrane interactions, elicits [Ca2+]i oscillations similar to those observed at fertilization [32, 35]. Furthermore, the [Ca2+]i oscillations generated by injection of soluble sperm extracts can support oocyte activation and/or development to the blastocyst stage in newly matured rabbit, mouse, cow, and pig oocytes [36–39], and when coinjected with either round spermatids or heat-inactivated sperm heads, both of which are incompetent to induce activation, can support development to term in the mouse [40, 41].

Therefore, we wanted to determine whether porcine sperm fractions (pSF) could also be used to activate and support the development of bovine nuclear transfer embryos. The objectives of this study were to determine 1) the optimum dose of pSF that triggers [Ca2+]i oscillations similar to those seen at fertilization and supports high rates of bovine oocyte activation and 2) the developmental potential of bovine nuclear transfer embryos activated by injection of pSF in comparison with nuclear transfer embryos subjected to chemical activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
In Vitro Maturation and Fertilization

Oocytes were obtained from 2- to 8-mm follicles from cow ovaries that had been collected at a slaughterhouse and transported back to the laboratory at 30°C. Oocytes with intact, compact cumulus cells were matured in TCM-199 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT), 0.1 U/ml of LH (Sioux Biochemical, Sioux Center, IA), and 1 µg/ml of estradiol. Oocytes were cultured in 300 µl in four-well tissue culture plates (Nunclon, VWR, Chicago, IL) for 20 h, and those oocytes to be used for microinjection or nuclear transfer procedures were stripped of cumulus cells by vortexing in Tyrode lactate (TL)-Hepes + 1 mg/ml of hyaluronidase for 5 min. Oocytes to be used for IVF were left intact and were washed several times in TL-Hepes and transferred to fertilization medium: 100 mM NaCl, 3 mM KCl, 0.27 mM CaCl2, 25 mM NaHCO3, 1 mM sodium lactate, 0.4 mM pyruvate, 1 mM L-glutamine, 6 mg/ml BSA (fatty acid free), 1% basal minimum essential (BME) amino acids, and 1% minimal essential medium (MEM) nonessential amino acids.

IVF was carried out as previously described [42]. Frozen semen (kindly donated by Genex, Ithaca, NY) was prepared according to the Percoll method, and motile sperm were added at a final concentration of 2.0 x 106 spermatozoa/ml. Capacitation was induced by adding 6 µg/ml of heparin to the fertilization medium [43]. Cumulus-intact oocytes were incubated with sperm in the fertilization medium for 18 h, after which they were stripped by vortexing in TL-Hepes for 2 min. Fertilized oocytes were then placed into 500 µl of ACM culture medium (100 mM NaCl, 3 mM KCl, 0.27 mM CaCl2, 25 mM NaHCO3, 1 mM sodium lactate, 0.4 mM pyruvate, 1 mM L-glutamine, 3 mg/ml BSA [fatty acid free], 1% BME amino acids, and 1% MEM nonessential amino acids) supplemented with 1% FCS in four-well plates containing mouse fetal fibroblasts and allowed to develop for different periods of time according to the experiment or used for Western blotting procedures. Embryos that were cultured to the blastocyst stage were transferred onto new mouse feeder layers supplemented with 10% FCS on Day 4 of development. Embryonic rates were recorded on Days 2 and 8 of development.

Sperm Factor Preparation

Cytosolic sperm fractions were prepared from boar semen as previously described [32, 44]. Semen samples were washed twice with TL-Hepes medium, and the sperm pellet was resuspended in a solution containing 75 mM KCl, 20 mM Hepes, 1 mM EDTA, 10 mM glycerophosphate, 1 mM dithiothreitol, 200 µM PMSF, 10 µg/ml pepstatin, 10 µg/ml leupeptin, pH 7.0 (all chemicals from Sigma, St. Louis, MO, unless otherwise specified). The sperm suspension was sonicated for 25–35 min at 4°C (XL2020; Heat Systems, Farmingdale, NY), the lysate was spun twice at 10 000 x g, and the supernatants were collected. The resulting supernatant was centrifuged at 100 000 x g for 1 h at 4°C, and the clear supernatant was used as the cytosolic fraction. Ultrafiltration membranes (Centriprep 10 and 30 and Centricon 30; Amicon, Beverly, MA) were used to wash the supernatant with injection buffer (75 mM KCl and 20 mM Hepes, pH 7.0). Crude sperm extracts were mixed with saturated ammonium sulfate to 50% saturation, the precipitates were collected by centrifugation (10 000 x g for 15 min at 4°C), and the pellets were stored at -20°C until use. The pellets were resuspended in injection buffer, washed several times in the same buffer, and concentrated with ultrafiltration membranes. Protein concentrations were determined using the bicinchoninic acid (BCA-1) assay for protein quantification.

Microinjection Techniques and [Ca2+]i Monitoring

Microinjection techniques were carried out as previously described [20]. Bovine oocytes were microinjected using Narishige manipulators (Medical Systems Corp., Great Neck, NY). Glass micropipettes were filled by suction of a microdrop containing either 0.5 mM fura-2 dextran (fura-2D, dextran 10 kDa; Molecular Probes, Eugene, OR) or pSF (1–15 mg/ml protein concentration). Solutions were injected into the cytoplasm of oocytes by pneumatic pressure (PL1-100, picoinjector; Harvard Apparatus, Cambridge, MA). Fura-2D fluorescence was monitored as previously described [20]. Excitation wavelengths were at 340 and 380 nm, and the emitted light was quantified by a photomultiplier tube after passing through a 500-nm barrier filter. Neutral density filters attenuated the intensity of excitation light, and the fluorescent signal was averaged for the whole oocyte. The [Ca2+]i concentrations were calculated as described previously [45, 46]; Rmin and Rmax values were 0.15 and 1.5, respectively. Monitoring of [Ca2+]i began 30–60 min following injection of fura-2D, 26–38 h after the onset of oocyte maturation. Oocytes were monitored individually in 40-µl drops of 1.25% sucrose in TL-Hepes on a glass coverslip on the bottom of a Petri dish covered with paraffin. Fluorescence ratios were determined every 8 sec for 30–180 min, depending on the experiment.

Oocyte Activation

Twenty-six to 28 h after the onset of maturation, bovine oocytes were injected with 1, 5, or 15 mg/ml pSF (concentration in the pipette), and immediately after completion of the injection, the oocytes were transferred back into culture medium. Control oocytes were injected with buffer alone, incubated in 5 µM calcium ionomycin (Calbiochem, San Diego, CA) for 4 min, or fertilized. Activation was assessed by the presence of pronuclear formation, which was evaluated 18 h following pSF injection or fertilization, and was visualized by labeling of the chromatin with bis-benzimide (Hoechst 33342) and examined by fluorescence microscopy in 70-µl drops of TL-Hepes under heavy mineral oil.

Western Blot Technique

Equal volumes of crude lysates from eight bovine oocytes and double-strength sample buffer [47] were combined as described previously [48]. Samples were boiled for 3 min and loaded into 4% SDS-polyacrylamide gels. The separated proteins were transferred onto nitrocellulose membranes (Micro Separation, Westboro, MA) using a Mini Trans Blot Cell (Bio-Rad Laboratories, Hercules, CA) for 2 h at 4°C. The membranes were first washed in PBS and 0.05% Tween (PBS-T) and then blocked in 6% nonfat dry milk in PBS-T for 1 h. After several washes in PBS-T, the membranes were incubated overnight with a rabbit polyclonal antibody raised against a 15-amino acid peptide sequence of the C-terminal end of the inositol 1,4,5-trisphosphate receptor 1 (IP3R-1) subtype (Rbt03) diluted to 1:3000 in PBS-T [49] (the antibody was a generous gift of Dr. J.B. Parys, University of Leuven, Leuven, Belgium). Following multiple washings, the membranes were incubated for 1 h with a 1:3000 dilution of a horseradish peroxidase-coupled secondary antibody. The membranes were developed using Western blot chemiluminescence reagents (NEN Life Sciences Products, Boston, MA) and exposed for 1–3 min to maximum sensitivity film (Kodak; Fisher Scientific, Springfield, NJ). Broad-range prestained SDS-PAGE molecular weight markers (Bio-Rad) were processed in parallel to estimate the molecular weights of the immunoreactive bands. The intensity of the IP3R-1 bands was quantified using Adobe PhotoShop (Adobe, Mountain View, CA) essentially as described previously [50] and plotted using Microsoft Excel (Microsoft, Redmond, WA). The mean pixel intensity within a selected set area containing the IP3R-1 band was obtained, and the same set area was applied to all lanes for that particular film. The same set area was also placed in an area of the film in which there were no bands, and a background number was obtained and subtracted from the previously obtained values. The band from MII oocytes was used as a reference and assigned the value of 1.

Nuclear Transfer

Nuclear transfer was carried out essentially as described previously [7]. At 20 h postmaturation (hpm), in vitro-matured oocytes were enucleated, and chromosome removal was confirmed by bis-benzimide staining under ultraviolet light. Enucleated oocytes were reconstructed with adult bovine fibroblasts from a cell line established from a 6-yr-old bull as described previously [7]. To obtain a synchronous population of G1 cells, 24 h prior to nuclear transfer fibroblasts were plated at a low density of 0.5 x 106 cells/100-mm tissue culture plate (Corning, VWR) in {alpha} MEM (Gibco) supplemented with 10% FCS, 0.15 g/ml glutamine, 0.003% ß-mercaptoethanol (Gibco), and antibiotic-antimycotic (MEM + FCS; Gibco). Twenty-four hours after the initial plating, G1 cells were isolated by briefly shaking the low-density plates for 30–60 sec at medium speed (Vortex-Genie 2; Fisher Scientific, Houston, TX). Cells actively dividing were easily detached and found in the supernatant. The supernatant culture medium was then collected and centrifuged at 500 x g for 5 min, and the resulting pellet was resuspended in 250 µl of TL-Hepes, which contains an enriched population of G1 cells [7]. G1 fibroblasts isolated in this manner were easily identified by the presence of a cytoplasmic bridge and were separated and transferred into the perivitelline space of MII oocytes. The reconstructed oocytes were placed in a fusion chamber formed by two wire electrodes located 0.5 mm apart, and fusion was carried out in 0.28 M sorbitol by administering an electrical pulse of 2.6 kV/cm2 for 20 µsec (Electro Cell Manipulator 200; Genetronics, San Diego, CA). Three to 4 h after fusion, 27–29 hpm, bovine oocytes reconstructed by nuclear transfer were injected with 5 mg/ml pSF and placed back into ACM culture medium containing 5.0 µg/ml of cytochalasin-B for 3–4 h to prevent extrusion of the second polar body. Alternatively, reconstructed oocytes were placed into 5 µM ionomycin for 4 min and then into 10 µg/ml of cycloheximide and 5.0 µg/ml cytochalasin-B for 5 h. Following activation, both groups of oocytes were washed 4 or 5 times in Hepes-buffered hamster embryo culture medium (114 mM NaCl, 3.2 mM KCl, 2 mM NaHCO3, 10 mM Hepes, and 1% BME amino acids) and placed in culture in 4-well tissue culture plates for 7–8 days with ACM culture medium + 1% FCS containing mouse fetal fibroblasts. Fifty embryos were placed in each well. On Day 4 of development, embryos were transferred onto new mouse feeder layers supplemented with 10% FCS. Embryonic development was recorded on Days 2 and 8.

Embryo Transfer and Pregnancy Detection

On Days 7 and 8 postactivation, grade 1 and grade 2 blastocysts resulting from nuclear transfer and IVF were transferred into Day 6 and Day 7 synchronized recipient cows. Either 1 IVF embryo or 2 nuclear transfer embryos were transferred into the uterine horn ipsilateral to the corpus luteum. Recipients were synchronized by a single 5-ml injection of Lutalyse (Pharmacia & Upjohn, Kalamazoo, MI) followed by estrus detection. Pregnancies were detected by ultrasound on Days 30, 60, and 90 and by rectal palpation on Days 120 and 150 of gestation.

Statistical Analysis

Statistical comparisons of the intensity of IP3R-1 bands and of Ca2+ parameters were performed using a Student t-test. To detect differences between different groups of oocytes subjected to different activation and nuclear transfer protocols, chi-square analysis was employed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
Effect of pSF Dose on [Ca2+]i Responses in Bovine Oocytes

To determine the dose of pSF that induces [Ca2+]i oscillations similar to those observed during fertilization, pSF with protein concentrations of 1, 5, or 15 mg/ml was injected into bovine oocytes at 26–30 hpm, and [Ca2+]i responses were monitored. Injection of 1 mg/ml pSF induced the first rise but failed to elicit the subsequent [Ca2+]i oscillations in 3 of 3 oocytes (Fig. 1A and Table 1), whereas injection of 5 mg/ml pSF elicited a first rise that was followed by [Ca2+]i oscillations that closely resembled fertilization-associated responses [20] with respect to amplitude and interval of [Ca2+]i rises in 6 of 7 oocytes (Fig. 1B and Table 1). Injection of 15 mg/ml pSF also elicited oscillations, but these responses exhibited an abnormally high frequency in all 5 oocytes (Fig. 1C and Table 1). Thus, 5 mg/ml pSF produced a [Ca2+]i response that most closely resembled the response observed during fertilization of bovine oocytes.



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  		FIG. 1. Representative [Ca2+]i profiles of bovine oocytes injected with different doses of pSF 26–28 h after the onset of maturation: 1 mg/ml (A), 5 mg/ml (B), and 15 mg/ml (C). Oocytes were monitored for 30–60 min following injection. Arrows denote the time of pSF injection


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  		TABLE 1. Characteristics of pSF-induced [Ca2+]i oscillations in bovine oocytes

Activation Response of Bovine Oocytes Injected with Different Concentrations of pSF

A physiological dose of pSF must produce results that resemble fertilization with respect to [Ca2+]i oscillations and must support high rates of oocyte activation. Therefore, it was of interest to determine the percentage of bovine oocytes matured for 26–28 h that exhibited activation, as judged by pronuclear formation 18 h after injection of 1, 5, or 15 mg/ml pSF. Oocytes were also fertilized, injected with buffer alone, or exposed to 5 µM ionomycin for 4 min (Table 2). Injection of 1 mg/ml pSF or exposure to 5 µM ionomycin induced poor rates of activation (Table 2; P < 0.05), whereas injection of 5 mg/ml pSF activated a percentage of oocytes that was comparable to IVF (Table 2; P > 0.05). In contrast, injection of 15 mg/ml pSF induced high rates of chromatin decondensation and/or fragmentation in oocytes and low rates of pronuclear formation. Thus, injection of 5 mg/ml pSF into bovine oocytes elicited both [Ca2+]i oscillations and activation responses that were similar to those observed at fertilization.


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  		TABLE 2. Activation of bovine oocytes injected with different concentrations of pSF

Effect of Oocyte Activation on In Vitro Development of Nuclear Transfer Embryos

To evaluate whether pSF could support activation and in vitro development of bovine nuclear transfer embryos, 5 mg/ml pSF was injected into oocytes reconstructed by nuclear transfer, and cleavage and development rates induced by this treatment were compared with those induced by chemical activation and by fertilization. This treatment induced high rates of cleavage and development in these embryos (Table 3). Nevertheless, chemically activated and IVF embryos exhibited higher rates of cleavage and development, and as expected, IVF embryos developed to the blastocyst stage at higher rates than reconstructed embryos regardless of the activation procedure (P < 0.05). Likewise, the rate of parthenogenetic development of bovine oocytes activated with chemicals was higher than that for oocytes activated by injection of pSF (P < 0.05; Table 3). Because embryos activated with pSF undergo an additional manipulation step (microinjection), we ran an injection control to test whether injection itself contributed to lower development. Injection of buffer followed by parthenogenetic activation with chemicals resulted in similar rates of development compared with uninjected controls, indicating that the injection procedure did not have a significant impact on development (data not shown). Furthermore, the proportion of grade 1 and grade 2 blastocysts resulting from nuclear transfer procedures was similar among activation treatments (data not shown).


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  		TABLE 3. In vitro development of bovine nuclear transfer (NT) embryos activated by pSF

In Vivo Development of Nuclear Trasfer Embryos Activated with pSF

To determine whether pSF could support in vivo development, resulting blastocysts from the nuclear transfer procedure were transferred into synchronized recipient cows, and development was monitored on Days 30, 60, 90, 120, and 150 of gestation. The development of nuclear transfer embryos activated chemically and of IVF embryos generated from the same oocytes was also tested (Fig. 2). At Day 30 of gestation, a similar number of pregnancies was observed among treatment groups (Fig. 2). However, between gestational Days 30 and 90, a large number of pregnancies were lost in both nuclear transfer groups (Fig. 2), and by 90 days of gestation no pregnancies remained in the pSF group. Likewise, control nuclear transfer embryos did not develop past 150 days of gestation. In contrast, only one of the pregnancies established following the transfer of IVF embryos was lost between 30 and 60 days of gestation, and the remaining five IVF embryos developed to term.



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  		FIG. 2. Developmental rates of nuclear transfer embryos activated with pSF in comparison to chemical activation and IVF on Days 30, 60, 90, 120, and 150 of gestation. Percentages represent the average of three to six replicates

Duration of [Ca2+]i Oscillations in Bovine Oocytes Activated with pSF

Because injection of pSF did not seem to improve the in vivo development of nuclear transfer embryos, we wanted to determine whether pSF was supporting [Ca2+]i oscillations for as long as they are observed during fertilization in bovine oocytes. To accurately assess the duration of the [Ca2+]i oscillations, individual oocytes were monitored at 0–1, 3–5, and 8–11 h following injection of 5 mg/ml pSF at 26–28 hpm (Fig. 3, A–C). Three of four oocytes injected with pSF supported [Ca2+]i oscillations as long as 3–5 h, and one of four oocytes exhibited oscillations for as long as 8 h. Thus, although injection of pSF into bovine oocytes appears to trigger the initiation of fertilization-like oscillations, these responses cease prematurely.



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  		FIG. 3. Duration of [Ca2+]i oscillations induced by an injection of 5 mg/ml pSF into bovine oocytes 26–28 h after the onset of maturation. Individual oocytes were monitored for 0–1 (A), 3–5 (B), and 8–11 (C) h following injection of pSF. The [Ca2+]i oscillations were induced to resume by a second injection of 5 mg/ml pSF 5–6 h after the first injection (D). Arrows indicate the time of the second injection in two separate oocytes

To determine whether pSF-injected oocytes were competent to support [Ca2+]i oscillations for an interval longer than that induced by a single injection, a second injection of 5 mg/ml pSF was administered 5 h after the first injection (Fig. 3D). The second injection of pSF induced a large first [Ca2+]i rise similar to the response observed following the first injection and also elicited [Ca2+]i oscillations in all four oocytes. Thus, oocytes injected with pSF are able to support long-term oscillations, although it appears that more than one injection of a physiological concentration of pSF is needed.

Downregulation of the IP3R-1 in Bovine Oocytes Injected with pSF

Downregulation of the IP3R-1 subtype, the Ca2+ release channel thought to mediate the majority of Ca2+ release at fertilization, occurs following fertilization [51, 52]. To determine whether pSF could induce downregulation of the IP3R-1 in bovine oocytes, injected oocytes were collected at 16 h postinjection, and the density of IP3R-1 was determined by Western blotting. Injection of 5 mg/ml pSF did not induce significant downregulation of IP3R-1 at the established time point (P > 0.05; Fig. 4), although strong downregulation of the receptor was observed in fertilized oocytes and in oocytes injected a second time with pSF at 16 h postactivation (P < 0.05). Therefore, a single injection of soluble pSF is not sufficient to downregulate IP3R-1 in bovine oocytes, and a second injection was required to simulate a fertilization-like response.



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  		FIG. 4. Western blot analysis and quantification of IP3R-1 in bovine oocytes injected with 5 mg/ml pSF (A and B). Eight oocytes were used in each lane. Untreated MII oocytes aged in vitro for 42 h were used as negative controls, and fertilized oocytes collected at 16 h postinsemination were used as positive controls. Oocytes were first injected with pSF 26 h after the onset of maturation, and 5 h later a second injection of pSF was administered. 1x and 2x denote one and two injections of pSF, respectively. Molecular weight standards (x10-3 of the actual) appear on the left. Treatments under bars with different letters denote significant differences (P < 0.05). Values represent the means of three to six Western blot experiments performed on different batches of oocytes


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
NOTE ADDED IN PROOF
 REFERENCES
 
Extensive work has been done on developing efficient methods for activating bovine oocytes used in nuclear transfer. Ethanol [53] and calcium ionophore [54], both of which induce a monotonic [Ca2+]i rise, have been used as activators in nuclear transfer, but these compounds are only effective in activating aged oocytes. More efficient methods for activating newly matured oocytes have been developed. 6-DMAP, a broad-spectrum serine/threonine phosphorylation inhibitor, in combination with ionomycin supports high rates of activation and embryonic development in bovine oocytes [16]. Likewise, cycloheximide, a nonspecific protein synthesis inhibitor, is also a very effective activating treatment when combined with an electrical pulse, ethanol, or calcium ionophore [7, 11, 17]. To date, there is no single activation agent available that can induce physiological [Ca2+]i oscillations capable of supporting development, unaided, in unaged bovine oocytes. Here, we demonstrate for the first time that a soluble factor extracted from porcine sperm can trigger physiological [Ca2+]i responses, initiate activation, and support fetal development in oocytes reconstructed by nuclear transfer.

As reported in previous studies, the [Ca2+]i responses and activation rates induced by injection of pSF in the present work were dose dependant [32, 36]. Injection of lower concentrations of pSF failed to trigger [Ca2+]i oscillations and a high percentage of oocytes arrested in metaphase III (MIII), a condition described previously in both recently ovulated mouse oocytes [55] and newly matured bovine oocytes [56] that occurs in response to insufficient Ca2+ stimulation. These results were similar to those observed in oocytes exposed to ionomycin, which also failed to activate and arrested in MIII. In contrast, injection of the highest concentration of pSF (15 mg/ml) triggered [Ca2+]i oscillations that were higher in frequency and lower in amplitude than those that are typically observed in fertilized bovine oocytes [20]. As a result of this response, most oocytes failed to properly activate and exhibited abnormal chromatin configurations. High-frequency [Ca2+]i oscillations induced by pSF have triggered abnormal activation in mouse oocytes [57], and our results in this study confirm those findings. Injection of 5 mg/ml triggered [Ca2+]i oscillations that resembled those associated with fertilization, and the majority of oocytes formed morphologically normal pronuclei. Collectively, these results demonstrate that doses of pSF that initiate physiological Ca2+ responses induce normal activation of bovine oocytes.

In spite of the success of the nuclear transfer technology, the efficiency of this procedure remains very low. Developmental rates for bovine nuclear transfer embryos in vitro to the blastocyst stage vary widely, with reported values ranging between 2% and 69% [15, 58], and the developmental rates obtained in the present study are similar to those previously reported. Multiple factors can contribute to the poor development of nuclear transfer embryos produced in vitro, such as oocyte quality, culture conditions, activation procedures, donor cell line, cell-cycle stage of the donor cell, reprogramming of the donor nucleus, and the lab technicians that are involved in reconstructing the nuclear transfer embryos. In this study, we assessed the impact of changing the activation conditions to more closely replicate sperm activation on the in vitro and in vivo developmental capacity of nuclear transfer bovine embryos. Despite our success of partially mimicking the sperm-triggered Ca2+ responses, which resulted in the ability of this treatment to induce development in unaged oocytes in the absence of supplementation with protein kinase and/or synthesis inhibitors, the developmental rates of reconstructed embryos activated by pSF remained low.

Although, one interpretation of these results is that activation protocols do not influence the outcome of nuclear transfer, there may be at least two factors that presently limit the effectiveness of pSF as a parthenogenetic agent for bovine oocytes. One problem may be the purity of the pSF preparation. The ability of sperm fractions to trigger Ca2+ release is highly conserved from frogs to humans [35, 59]. The active component(s) responsible for the Ca2+-releasing activity has not yet been identified, and only semipurified fractions are available. In our study, semipurified fractions were used to induce activation, and contaminant proteins, such as proteases from the acrosome, may be present in our fractions and may have a negative impact on embryo development. This potential problem can be remedied in the future by utilizing fractions of higher purity.

A second problem that may compromise the ability of pSF to support high rates of development is the delivery of the factor as a soluble component in a bolus injection. This method of release is unlikely to duplicate the gradual and persistent release thought to take place during fertilization. In bovine oocytes, rises in [Ca2+]i have been observed for as long as 22 h following the onset of fertilization and cease by the end of the first cell cycle [21]. In bovine oocytes injected with pSF, [Ca2+]i oscillations did not last more than 8 h, and in most oocytes, [Ca2+]i oscillations only lasted for 3–5 h. This short duration of the Ca2+ response induced by pSF did not affect pronuclear formation, although it may have had an impact on subsequent developmental rates. In mouse oocytes, [Ca2+]i oscillations have been shown to persist up to pronucleus formation, approximately 4 h after the onset of fertilization [60], and in these oocytes, pSF supports development to the blastocyst stage at rates similar to those obtained with fertilization [38] (unpublished results). The difference in efficiency of pSF as an activating agent in mouse and bovine oocytes may be due to the fact that the duration of [Ca2+]i oscillations induced by pSF in mouse oocytes closely resembles the duration of the sperm responses in this species but is too short for the longer cell cycle in bovine oocytes. In mouse oocytes, close correlations have been established among the persistence of [Ca2+]i oscillations, the number of inner cell mass cells, and implantation rates [61]. The [Ca2+]i oscillations may stimulate development by facilitating gene expression of developmentally relevant genes [29] or by promoting recruitment of maternal mRNAs and posttranslational modifications of proteins that may regulate early cleavage stages [62]. Hence, the inability of pSF to support long-lasting oscillations may compromise the ability of these embryos to reach full development.

The mechanism by which the sperm induces long-lasting Ca2+ release following fertilization is currently not known, although there is evidence to indicate that the oocyte-activating component in mouse sperm may be associated with the sperm's perinuclear theca. For instance, injection of sperm heads pemeabilized with Triton-X, a mild detergent, are still able to trigger Ca2+ release [63] and activate oocytes [41], whereas combined treatment with the reducing agent dithiothreitol abolishes the mouse sperm's ability to induce activation, probably through solublization of the active factor from the perinuclear theca [41]. Remarkably, the sperm's Ca2+ releasing component appears to associate with the pronucleus soon after fertilization [64, 65], as indicated by the ability of karyoplasts from zygotes to initiate [Ca2+]i oscillations upon transfer into unfertilized oocytes [65]. Nonetheless, it is presently not known whether the putative activating factor is a component of the perinuclear theca or binds to it through an intermediary protein and whether this interaction modulates its release during fertilization and subsequent relocation to the pronuclear membranes. In the present study, the premature cessation of pSF-induced [Ca2+]i oscillations may be due to the lack of this plausible association/stabilizing factor, the absence of which may leave the active protein susceptible to premature degradation or inactivation.

The limited persistence of oscillations by pSF was further confirmed by the findings that a single injection of pSF induced very poor downregulation of the IP3R-1. The IP3R-1 is the predominant isoform expressed in mouse and bovine oocytes [51, 52]. In these oocytes, a marked reduction in the IP3R-1 immunoactivity occurs during fertilization and appears to be associated with production of inositol 1,4,5-trisphosphate (IP3), because it was not observed following parthenogenetic activation with compounds that do not activate the phosphoinositide pathway, such as ethanol, ionomycin/6-DMAP, or strontium chloride [66]. Injection of adenophostin A [67], a potent agonist of the IP3R, into mouse and bovine oocytes [52, 66] or of pSF into mouse oocytes [66] significantly downregulated the receptor. In bovine oocytes, a single injection of pSF failed to induce downregulation of the IP3R-1, and the limited duration of pSF activity in bovine oocytes appears responsible because a second injection of pSF 5 h following the first injection induced significant downregulation of IP3R-1. Thus, in bovine oocytes multiple injections of pSF may be necessary to support long-lasting [Ca2+]i oscillations and physiological activation, although multiple injections may be technically difficult.

We demonstrated that pSF can effectively trigger the initiation of development in reconstructed bovine oocytes. However, its efficacy may be limited because of premature cessation of the induced oscillations. Future research will focus on the generation of highly purified fractions that may support the generation of [Ca2+]i oscillations for an extended period of time in bovine oocytes.


   NOTE ADDED IN PROOF
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
REFERENCES
 
Following the completion of this manuscript, two cloned calves were born following nuclear transfer using pSF as the activating method.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Tara L. King, Karli J. Wright, and Warren S. Lucero for their technical assistance and Audy R. Spell for embryo transfer. We also thank Jeremy Smyth for critically reviewing this manuscript.


   FOOTNOTES
 
First decision: 18 September 2001.

1 This work was supported by USDA grants (97-2919, 99-2371) to R.A.F. Back

2 Correspondence: James M. Robl, Hematech LLC, 4 Biotech Park, 377 Plantation Dr., Worcester, MA 01605. FAX: 508 792 0782; jrobl{at}hematech.com Back

Accepted: November 7, 2001.

Received: August 16, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 

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