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BOR - Papers in Press, published online ahead of print March 19, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.012823
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BIOLOGY OF REPRODUCTION 69, 186–194 (2003)
DOI: 10.1095/biolreprod.102.012823
© 2003 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Cytoskeleton and Chromatin Reorganization in Horse Oocytes Following Intracytoplasmic Sperm Injection: Patterns Associated with Normal and Defective Fertilization

Jordi L. Tremoleda1,2, Theo van Haeften3, Tom A. E. Stout2, Ben Colenbrander2,4, and Mart M. Bevers4

Departments of Equine Sciences,2 Biochemistry, Cell Biology, and Histology,3 Farm Animal Health,4 Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracytoplasmic sperm injection (ICSI) is the method of choice for fertilizing horse oocytes in vitro. Nevertheless, for reasons that are not yet clear, embryo development rates are low. The aims of this study were to examine cytoskeletal and chromatin reorganization in horse oocytes fertilized by ICSI or activated parthenogenetically. Additional oocytes were injected with a sperm labeled with a mitochondrion-specific vital dye to help identify the contribution of the sperm to zygotic structures, in particular the centrosome. Oocytes were fixed at set intervals after sperm injection and examined by confocal laser scanning microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle and the first polar body. After sperm injection, an aster of microtubules formed adjacent to the sperm head and subsequently enlarged such that at the time of pronucleus migration and apposition it filled the entire cytoplasm. During syngamy, the microtubule matrix reorganized to form a mitotic spindle on which the chromatin of both parents aligned. Finally, after nuclear and cellular cleavage were complete, the microtubule asters dispersed into the interphase daughter cells. Sham injection induced parthenogenetic activation of 76% of oocytes, marked by the formation of multiple cytoplasmic microtubular foci that later developed into a dense microtubule network surrounding the female pronucleus. The finding that a parthenote alone can produce a microtubule aster, whereas the aster invariably forms at the base of the sperm head during normal fertilization, indicates that both gametes contribute to the formation of the zygotic centrosome in the horse. Finally, 25% of sperm-injected oocytes failed to complete fertilization, mostly due to absence of oocyte activation (65%), which was often accompanied by failure of sperm decondensation. In conclusion, this study demonstrated that union of the parental genomes in horse zygotes is accompanied by a series of integrated cytoskeleton-mediated events, failure of which results in developmental arrest.

fertilization, in vitro fertilization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The commercial application of in vitro embryo production (IVP) to horses has been limited by the poor rates of fertilization obtained after conventional in vitro fertilization (IVF) [1–3], which appears to be primarily due to the poor capacity of stallion spermatozoa to penetrate the zona pellucida in vitro [4, 5]. Recently, intracytoplasmic sperm injection (ICSI) was introduced as an alternative to conventional IVF and has resulted in the birth of several foals [6–10]. By bypassing the critical events of oocyte penetration, ICSI has proven to be a promising means of producing equine embryos in vitro. There are, however, large differences in the rates of successful fertilization reported by different groups, with the rate of male pronucleus formation in horse oocytes fertilized by ICSI ranging from 21% to 71% [11–14] and cleavage rates ranging from 20% to 80% [9–12, 14]. These differences indicate that significant failures in zygote development still occur despite injection of a motile sperm into a mature oocyte. In addition, because in vitro culture conditions are still not optimal, only a low percentage of cultured zygotes develop to the blastocyst stage (1% to 30% of cleaved oocytes) [11, 13, 15, 16]. At present, transferring ICSI-derived horse zygotes into the oviduct of a ewe remains the most successful method for producing horse blastocysts ex vivo, with rates reaching 50% of cleaved oocytes [10]. Moreover, most equine ICSI pregnancies have resulted from the transfer of early zygotes to the oviduct of a recipient mare [6–8], which underlines the negative effects of in vitro culture on the developmental capacity of equine ICSI-produced zygotes.

Although ICSI can be applied successfully to horses, the disparity in fertilization rates and the inefficiency of current culture conditions remain obstacles to the development of a reliable and reproducible system for producing horse embryos in vitro. To improve the efficiency of equine IVP, it is important to gain more insight into the processes of fertilization and embryo development after sperm penetration in vivo. Since in vivo embryos are retained within the fallopian tube until Day 5–6 after ovulation, from where they can only be recovered by surgery or slaughter of the mare, only limited information is available on the sequence of events that occurs during fertilization and early equine embryonic development in vivo. Enders et al. [17] reported clear signs of fertilization as early as 10 h after mating in oviductal zygotes, when the incorporated sperm had acquired its pronuclear envelope and the oocyte had progressed to telophase of the second meiotic division. By 12 h after mating, horse zygotes had reached the pronuclear stage [1, 17, 18]. Similarly, because of the difficulties of producing equine zygotes in vitro and since most zygotes are transferred to the oviduct of a recipient as soon as possible after cleavage, only a few studies on embryo development in culture have been documented. These studies described the development in culture of horse oocytes fertilized in vivo and recorded the first cell division to occur between 22 and 24 h after ovulation [1]. More recently, sperm chromatin decondensation has been reported to occur between 2 and 4 h after IVF [19] or ICSI [13, 14], with pronucleus formation following at around 16–24 h after sperm incorporation.

In general, for fertilization to proceed, a series of cytoplasmic and nuclear changes must occur in a precisely orchestrated fashion. Changes in nuclear structure include the formation of the male and female pronuclei (FPNs), migration and apposition of these pronuclei, and mixing of the maternal and paternal genomes. Finally, initiation of a mitotic division heralds the onset of embryonic development (reviewed by Yanagimachi [20]). These nuclear events are, however, highly dependent on reorganization of the microtubular and microfilamentar elements of the fertilizing gametes. An oocyte loses its centrosome, the organelle that acts as a microtubule organizing center, early in gametogenesis. During fertilization, it is the sperm centrosome that acts as the zygotic microtubule organizing center and induces formation of the radial microtubule-containing structure, the sperm aster, that coordinates migration and union of the two pronuclei and formation of the mitotic spindle (reviewed by Schatten [21]). This paternal inheritance of the centrosome is seen in most mammalian species studied, including humans [22], nonhuman primates [23], and many domestic animal species (e.g., cow [24], pig [25], sheep [26]). In contrast, the centrosome in rodent zygotes has a maternal origin [27, 28]. Little is known about the pattern of centrosomal inheritance and subsequent cytoskeletal dynamics in the horse zygote during fertilization.

The aim of this study was to characterize the nuclear and cytoskeletal events that occur in horse oocytes during fertilization after ICSI by examining chromatin, microtubule, and microfilament organization in oocytes cultured for up to 48 h after sperm injection. In addition, we investigated the stages in which the fertilization process deviates in cases of arrest or delay of zygote development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection and Culture of Cumulus Oocyte Complexes

The procedures for collecting and culturing horse oocytes have been described previously [29]. In brief, cumulus-oocyte complexes (COCs) were recovered by aspiration from the ovaries of slaughtered mares within 3–5 h after slaughter during the breeding season (March–September). All visible follicles less than 30 mm in diameter were punctured, and the follicular lumenae were flushed with PBS supplemented with 50 mg/L of BSA (Sigma, St. Louis, MO) and 25 IU/ml of heparin (Leo Pharmaceutical, Weesp, the Netherlands). The ovary was then bisected and any exposed follicles were similarly aspirated and flushed. Next, COCs were isolated by examining the collected fluid with a stereomicroscope. Recovered COCs were evaluated for their quality and washed twice in Hepes-buffered Tyrode medium containing 0.1% (w/v) polyvinylalcohol and 0.2% BSA (Hepes-TL-PVA). Oocytes with a complete, compact, multilayered cumulus were selected and washed twice with maturation medium before being placed in the culture medium proper. In our experimental protocol, we used only compact COCs because we considered them to be a more homogeneous population than the expanded COCs, which displayed different degrees of expansion within the granulosa, cumulus, and corona cells. Finally, oocytes were allocated randomly into groups of 20–25, which were incubated for 36 h in 500-µl aliquots of M199 supplemented with 10% fetal calf serum and 0.01 U/ml of both porcine FSH and equine LH (both Sigma) at 39°C in a humidified atmosphere of 5% CO2 in air.

Preparation of Spermatozoa for ICSI

Ejaculated sperm from a stallion of proven fertility was used for ICSI after freezing and thawing in the manner described by Parlevliet et al. [30]. The straws were thawed at 37°C for 45 sec, and the spermatozoa were rinsed free of cryoprotectant by centrifugation at 700 x g for 15 min in Hepes-buffered modified Tyrode medium (Sp-TALP; Parrish et al. [31]). The resuspended sperm pellet (approximately 200 µl) was then placed at the bottom of a 10-ml tube containing 2 ml of Hepes-Sp-TALP and incubated at 39°C in an atmosphere of 5% CO2 in air for "swim-up." After 40 min, the uppermost 1.5 ml of medium was collected and centrifuged at 400 x g for 5 min in a 2-ml polypropylene tube. The sperm pellet was again resuspended in 1 ml of Hepes-Sp-TALP, and the suspension was maintained at 38°C until ICSI. No attempt was made to induce sperm capacitation, although the bicarbonate in Sp-TALP medium should have stimulated this process [32].

Preparation of Oocytes and ICSI

After 36 h of in vitro maturation, the COCs were incubated for 5 min at 37°C in calcium- and magnesium-free Earle balanced salt solution (Gibco BRL, Paisley, UK) containing 0.1% (w/v) hyaluronidase (type I-S; Sigma H-3506). Next, the cumulus cells were removed by aspirating the COCs several times through a fine pipette or, if necessary, by vortexing. Cumulus-free oocytes were examined with an Olympus SZX9 (Olympus, Tokyo, Japan) inverted microscope (x400), and those with an intact oolema and an extruded first polar body were selected for ICSI. The selected oocytes were washed twice in Hepes-buffered synthetic human tubal fluid (Q-HTF Hepes; BioWhittaker, Verviers, Belgium) supplemented with 0.4% BSA and maintained in synthetic human tubal fluid (Q-HTF; BioWhittaker) containing 0.4% BSA until injection. Oocyte injection was performed in a 5-µl microdrop of Q-HTF Hepes at 37°C on a heated stage (Linkam Scientific Intruments, Tadworth, UK) mounted on an Olympus-CK40 inverted microscope equipped with Narishige micromanipulators (Narishige Co., Ltd., Tokyo, Japan). Sperm injection was performed essentially in the same manner as described by Palermo et al. [33]. Just before injection, 2 µl of the motile sperm suspension was mixed with 5 µl of clinical grade polyvinylpyrrolidone (PVP) (10% PVP in Hepes-buffered salt solution; Lucron Bioproducts B.V., Gennep, the Netherlands) to slow sperm movement and aid capture. A motile spermatozoon was then immobilized by swiping the injection pipette (10-MIC-Angled 30°; Gynotec, Malden, the Netherlands) across its tail, and it was then moved, with a minimal volume of medium, to the microdrop containing the oocyte. For sperm injection, an oocyte was held stationary by suction via the holding pipette (10-MPH-120-Angled 40°; Gynotec) with the polar body positioned at 6 or 12 o'clock. The injection pipette containing the spermatozoon was advanced through the zona pellucida and plasma membrane at the 3 o'clock position, and the spermatozoon was injected into the ooplasm with a minimal volume of accompanying medium. Further oocytes were sham injected with PVP solution only. After injection, the oocytes were returned to the Q-HTF medium, and within 15–30 min they were transferred to 20-µl drops of fresh Q-HTF medium containing 0.4% BSA and cultured at 39°C in 5% CO2 in humidified air.

Experimental Design

To describe the chronology of cytoskeleton and chromatin rearrangements in horse oocytes during fertilization induced by ICSI, injected oocytes were cultured for 24 or 48 h and then labeled simultaneously with stains for chromatin, microfilaments, and microtubules. To further characterize the events associated with sperm reorganization within the activated oocyte and the changes in microtubule distribution that accompany pronucleus formation, additional injected oocytes were cultured for 6, 12, or 18 h before being stained for the visualization of microtubules and chromatin. To track sperm incorporation and the conversion of sperm-derived structures into zygotic structures, oocytes were injected with a sperm labeled with a mitochondrion-specific vital dye [34]. Finally, the organization of microtubules and chromatin in sham-injected oocytes was examined to elucidate the role of oocyte-derived structures in maternal pronucleus formation and oocyte activation.

Immunocytochemistry and Confocal Laser Scanning Microscopy

Visualization of microtubules, microfilaments, and chromatin in horse zygotes After 6, 12, 18, 24, or 48 h, the presumptive zygotes were removed from culture and permeabilized by incubating them for 15 min in buffer M, a glycerol-based, microtubule-stabilizing solution, at 37°C [35]. The zygotes were then fixed in 3% paraformaldehyde in PBS and subsequently maintained in the fixative at 4°C for 4–6 days before staining. Following fixation, the zygotes were washed twice in PBS containing 150 mM glycine and 0.1% (w/v) BSA (both Sigma) for 15–30 min to reduce free aldehydes and to block nonspecific reactions. Depending on the duration of the incubation, injected oocytes were double (6-, 12,- and 18-h incubations) or triple (24- and 48-h incubations) stained using different combinations of fluorescent probes. In all cases, microtubules were labeled first by incubating the zygotes with a monoclonal anti-{alpha}-tubulin antibody (T-5168; Sigma) diluted 1:250 in PBS containing 0.5% (v/v) Triton X-100 and 0.1% BSA (PBS-TX100-BSA) for 90 min at 37°C. Next, the zygotes were washed three times in PBS-TX100-BSA before being incubated for 1 h in a blocking solution (0.1 M glycine, 1% goat serum, 0.01% Triton X-100, 0.5% BSA, and 0.02% sodium azide; all from Sigma). The zygotes were then incubated for 1 h at 37°C in goat anti-mouse antibody diluted 1:100 in PBS-TX100-BSA and conjugated to either AlexaFluor 488 (A-11029; Molecular Probes Europe BV, Leiden, the Netherlands) or tetramethylrhodamine isothiocyanate (TRITC; T-5393; Sigma) for the dual and triple stained zygotes, respectively. Once their microtubules had been labeled, the zygotes were washed once with PBS-TX100-BSA and twice with PBS alone. Next, the presumptive zygotes from the 24- and 48-h cultures were incubated for 1 h with AlexaFluor 488 Phalloidin (15 IU/ml; A-12379; Molecular Probes) to enable microfilament detection. Injected oocytes from 6-, 12-, and 18-h cultures were not stained for microfilament detection. Finally, to enable visualization of the DNA, the presumptive zygotes from 24- and 48-h cultures were incubated with TO-PRO3 (5 µM in PBS; T-3605; Molecular Probes) for 15 min, whereas those from 6-, 12-, or 18-h cultures were stained for 15 min with Ethidium homodimer (EthD-1; 2 µM in PBS; E-1169; Molecular Probes).

Tracking sperm chromatin within the zygote To differentiate the paternal and maternal chromatin and to describe their contribution to zygotic structures during ICSI-induced fertilization, injected sperm cells were tagged with a mitochondrion-specific vital dye. For this, frozen/thawed ejaculated stallion sperm were selected by swim-up in Sp-TALP medium, as described previously. The motile sperm fraction was recovered and centrifuged at 400 x g for 5 min, and the resulting sperm pellet was resuspended in a 500 µM solution of Mitotracker Red (CMH 2XROS; M-7512; Molecular Probes) in Sp-TALP, in which it was incubated for 30 min at 39°C in 5% CO2 in air. The labeled sperm were then washed by two cycles of centrifugation and resuspension in Sp-TALP medium. ICSI with tagged sperm was performed as described previously, and within 10 min after ICSI, the injected oocytes were transferred to the incubation droplets. After a 6-h incubation, the developing zygotes were removed from culture, fixed in 3% paraformaldehyde in PBS, and maintained in the dark at 4°C for 1–2 days. Fixed zygotes were permeabilized by incubation for 30 min at room temperature in PBS containing 0.1% Triton X-100 and 0.1% BSA, and possible nonspecific labeling was prevented by incubation for 1 h with the blocking solution described herein. Localization of labeled microtubules was performed using a mouse monoclonal anti-{alpha}-tubulin antibody (Sigma) diluted 1:250 in PBS-TX100-BSA and the goat anti-mouse secondary antibody, this time conjugated to AlexaFluor 633 (1:100 in PBS-TX100-BSA; A-22284; Molecular Probes). DNA was stained with SYTOX Green (1 µM in PBS; S-7020; Molecular Probes).

Confocal laser scanning microscopy Stained presumptive zygotes were mounted on glass slides with an antifade-containing mounting medium (Vectashield; Vector Lab, Burlingame, CA). To avoid excessive pressure being exerted on the mounted oocytes, coverslips were supported by thick droplets of a Vaseline-wax mixture placed at each corner and sealed with nail polish. These zygotes were examined using a laser scanning confocal microscope (Leica TCS MP; Leica, Heidelberg, Germany) attached to an inverted microscope (Leica DM IRBE) equipped with 40x and 100x oil immersion objectives. The laser scanning confocal microscope was equipped with three lasers (Krypton 563 nm, Argon 514 nm, and HeNe 633 nm) for the simultaneous excitation of Alexa Fluor 488 or SYTOX Green, TRITC, EthD-1 or Mitotracker Red, and TO-PRO3 or AlexaFluor 633 using 488/568/650 nm excitation/barrier filter combinations. To avoid cross-talk of the acquired images in the photomultiplier channels, specimens were scanned using a sequential scanning mode. Images were recorded digitally and processed using Adobe Photoshop 5.5 software (Adobe Systems Inc., Mountain View, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 371 in vitro-matured horse oocytes with a first polar body and an intact oolemma were subjected to ICSI and cultured for various intervals. By the time of confocal laser scanning microscopy (CLSM) analysis after culture, however, 24 (6%) of the oocytes displayed completely aberrant and uninterpretable chromatin and cytoskeletal patterns and were therefore considered to be degenerate and were excluded from further analysis. In total, 347 oocytes (94%) were analyzed after ICSI by CLSM imaging (Fig. 1), and a further 60 oocytes were analyzed after sham injection.



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  		FIG. 1. Graphic representation of the dynamics of cytoskeleton and chromatin reorganization in horse oocytes fertilized by ICSI and incubated for a further 6–48 h. The series of cellular changes that occurred during fertilization have been categorized as A) oocyte activation and/or sperm decondensation; this encompasses the events beginning with the progression of oocytes from arrest in metaphase II of meiosis to formation of a FPN and decondensation of sperm chromatin to eventual formation of the male pronucleus; B) pronucleus formation, including migration and apposition under the direction of the sperm aster; and C) cellular cleavage, including all fertilized oocytes that progressed to the two-cell stage or beyond. The number of oocytes analyzed at each time point is shown above each column. Oocytes that failed to complete fertilization were excluded from the figures

Cytoskeleton and Chromatin Organization in Horse Oocytes after ICSI

In oocytes examined shortly after injection and while still arrested at metaphase II, microtubules were detected in the meiotic spindle and the first polar body only, as reported previously [29]. At this stage, the spindle appeared as a barrel-shaped conglomeration of microtubules with two anastral poles and with the chromosomes aligned along the meiotic plate (Fig. 2A). The polar body appeared as an amorphous mass of microtubules intertwined with chromatin (Fig. 2A and B). At 6 h after injection, densely stained microtubules could be observed radiating from the base of the decondensing sperm head (Fig. 2B and C). As the sperm chromatin continued to decondense to form the male pronucleus, the microtubules elongated further to form the radial sperm aster that had a distinct nucleation site at the sperm centrosome (Fig. 2I). Most oocytes displayed signs of activation 6 h after sperm injection (75%, 41/55; Fig. 1). During activation, the maternal chromosomes, which were initially compact and aligned along the meiotic plate (Fig. 2A), began to enlarge (Fig. 2D), and, thereafter, the oocyte proceeded through anaphase (Fig. 2E) and entered telophase (Fig. 2F) of the second meiotic division. This resulted in the formation of the second polar body; at this point, the microtubules were still visible in the meiotic midbody between the newly formed polar body and the developing FPN (Fig. 2F). After 12 h of incubation, 50% (18/36; Fig. 1) of the injected oocytes had reached the pronuclear stage, whereas the remainder of the cells were still in earlier stages of activation and sperm decondensation. At 18 h after injection, most of the presumptive zygotes (74%, 23/31; Fig. 1) had reached the pronuclear stage. During the development of pronuclei, the sperm aster continued to enlarge until it filled the entire cytoplasm (Fig. 2G). The sperm aster was not orientated preferentially toward the female nucleus but instead assumed a perinuclear distribution around both parental pronuclei, and, at this stage, no distinct nucleation sites were visible (Fig. 2H). By 18 h, the male and FPNs had enlarged and, presumably assisted by the sperm aster, had migrated to become apposed in an eccentric position within the cytoplasm, with an extremely dense array of microtubules between them (Fig. 2I). At 24 h after injection, pronuclear apposition was still the dominant feature of most zygotes (55%, 24/44; Fig. 1), although they had proceeded further toward syngamy, with the microtubules now concentrated at the poles of the adjacent pronuclei. In addition, a small number of zygotes had entered the first mitotic metaphase (5%, 2/44) during which the microtubule array developed into a bipolar structure that formed the mitotic spindle and held the now condensed chromatin along the mitotic plate. Although some two-cell embryos were detected as early as 6 h after injection, it was not until 48 h that a significant proportion of the injected oocytes (36%, 35/96; Fig. 1) had undergone cellular cleavage. The resulting two- to four-cell embryos had most of their microfilaments concentrated in the cell cortex, and, sometimes, a distinct microfilamentar cleavage furrow was visible at the intercellular junction (Fig. 2J and K). The microtubules were organized in a network that spread throughout the cytoplasm of the daughter cells but was particularly prominent around the decondensed chromatin of the interphase nuclei (Fig. 2L).



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  		FIG. 2. Laser scanning confocal images of horse oocytes during fertilization and early embryonic development after ICSI. In each case, microtubules are represented in green, microfilaments in blue, and chromatin in red. Shortly after ICSI, microtubules were seen in the meiotic spindle of the metaphase II oocyte or in the first polar body (A) and alongside the incorporated sperm (B). Metaphase plate (MP); polar body (PB). The sperm head began to decondense while still attached to the tail (B). The sperm aster formed as a microtubular array nucleating from the base of the decondensing sperm head, which would later develop into the male pronucleus (C). In metaphase II-arrested oocytes, microtubules were confined to the polar body and to the spindle, which held the maternal chromosomes along the meiotic plate. Sperm injection initiated oocyte activation, which was characterized by a resumption of meiosis during which the maternal chromosomes enlarged (D) and begin to migrate along the spindle toward the poles (E). At the telophase stage, the astral microtubules were found between the decondensing sets of female chromosomes (F). During the formation of the sperm aster, a distinct microtubule nucleation site (arrow) was detected adjacent to the sperm head, and during pronuclear migration, this array of microtubules expanded to fill the whole cytoplasm until it formed a microtubule matrix without a distinct nucleation site and surrounding both the male pronucleus and FPN (G, H). During pronuclear apposition before syngamy, a dense microtubule array (arrow) without a distinct bipolar centrosomal appearance was detected between the male pronucleus and FPN (I). In two- and four-cell embryos, the microfilaments were concentrated in the cortex of the daughter cells and at the cleavage furrows (arrow) (J, K). Microtubules formed a network surrounding the interphase nucleus of the daughter cells and extending throughout the cytoplasm (L). Microfilaments (MF); microtubules (MT). GBar = 6 µm (A, D, E, and F), 10 µm (B, C, and L), and 20 µm (G–K)

Microtubule and DNA Patterns in Sham-Injected Oocytes

Of the 60 metaphase II-stage oocytes examined by CLSM 24 h after sham ICSI, 13 (22%) showed no apparent signs of activation or further development; their microtubules remained concentrated in the second meiotic spindle and first polar body, and their chromosomes remained aligned at the metaphase plate (Fig. 2A). In 43% (26/60) of sham-injected oocytes, multiple microtubule arrays were detected, distributed randomly throughout the cytoplasm (Fig. 3A). In addition, although most meiotic spindles remained in the intact metaphase II form, in some oocytes the microtubular spindle was wider than normal and the meiotic plate seemed to have begun reorganization, a change usually associated with the early stages of oocyte activation. Seventeen percent of sham-injected oocytes (10/60; Fig. 3B and C) displayed more advanced features of activation, including microtubule aster assembly and formation of a microtubule network extending throughout the cytoplasm and accompanied by a resumption of meiosis and progression to telophase II. FPN formation was observed in 13% (8/60) of the sham-injected oocytes as a mass of decondensed chromatin surrounded by a network of disarrayed microtubules (Fig. 3D). In two cases (3%), the sham-injected oocytes developed gynogenetically with the second polar body remaining within the oocyte to form a second FPN.



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  		FIG. 3. Laser scanning confocal images of horse oocytes during parthenogenesis induced by sham injection (A–D) and sperm-injected oocytes that failed to progress through fertilization (E–L). A) In early horse parthenotes, microtubules were present in the metaphase II spindle, the polar body (PB), and multiple foci distributed throughout the cytoplasm (A). Metaphase plate (MP). These microtubule foci extended and coalesced to form a dense meshwork of microtubules extending from the remnants of the meiotic spindle, while the chromosomes began to decondense (B, C). Activated metaphase plate (Ac-MP). In this parthenote, a distinct FPN was seen surrounded by a dense network of disarrayed microtubules (D). The most common recorded cause of fertilization failure after ICSI was defective oocyte activation (E, F). The oocytes failed to resume meiosis and retained an intact metaphase plate, although the maternal chromosomes often became dispersed along the spindle away from the meiotic plate (G). In addition, the sperm chromatin frequently remained condensed in oocytes that failed to activate (F). In some cases of failed fertilization, multiple microtubule asters (arrow) were detected in the cytoplasm of injected oocytes, presumably originating from fragmentation of a defective meiotic spindle (H, I). The enlargement from the figure (I) shows microtubular threads detaching from the spindle. Further defective oocytes displayed two meiotic spindles due to a premature condensation of the sperm chromatin after ICSI (J) or three pronuclei (Digyny) due to failure to extrude the second PB, which was retained as a second FPN (K, L). Arrow indicates the microtubule domain concentrating between the pronuclei. Bar = 20 µm (A–E, H, I, L), 10 µm (F, G, and J), and 6 µm (I, inset)

Fertilization Failures and Developmental Arrest in Horse Zygotes after ICSI

Of the 347 oocytes injected, 262 (76%) displayed cytoskeletal and chromatin patterns consistent with normal fertilization; the remaining 85 oocytes (24%; see Fig. 4 for the effect of incubation time) showed signs of failed fertilization or developmental arrest. Since no reports are available on fertilization failure after ICSI in the horse, the findings were described according to the sequence of events reported herein (summarized in Fig. 6) and by comparison with the abnormalities of fertilization reported following ICSI in humans [36–38]. Most injected oocytes that failed to complete fertilization did not even reach the pronuclear stage. Indeed, most defects (88%, 75/85; Fig. 5) were associated with aberrant sperm integration and failure of the oocyte to complete meiosis after sperm incorporation. In total, 55 of the 85 fertilization failures (65%; Fig. 5) were characterized by the inability to progress properly through the second meiotic division. Affected oocytes displayed either an intact meiotic spindle (Fig. 3E and F) or a defective spindle with disorganized microtubules and the chromosomes displaced from the meiotic plate (Fig. 3G). In several of these oocytes, the sperm chromatin remained condensed or only partially decondensed (Fig. 3F), presumably due to failure of the sperm plasma, acrosomal, or nuclear membranes to break down. Another occasional defect was the presence of disarrayed microtubules in the cytoplasm of the injected oocyte (Fig. 3H and I). Such multiple microtubule nucleation centers were detected in 12% (10/85; Fig. 5) of arrested oocytes and seemed to arise from fragmentation of a defective meiotic spindle, particularly in those oocytes that remained arrested in metaphase II (Fig. 3I and inset). On rare occasions, two spindles were detected in the injected oocytes (6%, 5/85; Fig. 3J). These oocytes had failed to resume meiosis, and it is assumed that the paternal chromosomes had instead condensed prematurely to form a paternal meiotic spindle. Those oocytes that did reach the pronuclear stage but failed to enter the mitotic cycle were all found to possess three pronuclei and a weakly stained first polar body, with only one of the pronuclei associated to a sperm tail (abnormal tripronuclear zygote; Digyny: 12%, 10/85; Fig. 3K and L). Finally, in 5 (6%) of the 85 defective zygotes, no sperm was detected and the only DNA visible was that present in the meiotic spindle and in the first polar body (as in Fig. 2A).



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  		FIG. 4. The proportion of sperm-injected, in vitro-matured horse oocytes progressing normally or showing signs of defective fertilization 6, 12, 18, 24, and 48 h after ICSI. The different stages of fertilization were identified by CLSM analysis of cytoskeletal and chromatin configurations and based on the sequences described in Figure 1. Oocytes that displayed obviously abnormal cytoskeletal or chromatin patterns were considered to have suffered fertilization failure (defective or arrested oocytes). By 24 and 48 h after injection, a higher proportion of ICSI-derived oocytes showed signs of progression through normal fertilization that at 6, 12, or 18 h (*P < 0.05, chi-square test). Nevertheless, a significant proportion of those fertilized oocytes had not progressed beyond the early events of oocyte activation and/or sperm decondensation despite the time after sperm injection. However, since no abnormalities could be identified in their cytoskeletal or chromatin patterns, those oocytes were categorized as fertilized rather than arrested.



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  		FIG. 6. A schematic representation of the microtubule and chromatin reorganizations that occur in horse oocytes fertilized by ICSI. A mature oocyte is injected while arrested in metaphase of the second meiotic division, when it contains a second meiotic spindle and a first polar body. After sperm injection, a microtubule aster forms at the base of the decondensing sperm head and the arrested oocyte is activated, progresses through meiosis, and forms the second polar body. Annexation of the second polar body from the zygote is aided by microtubules aligned at the midbody of the second meiotic spindle. As the male pronucleus and FPN continue to decondense, the sperm aster enlarges to assist their migration and apposition. The microtubule aster that surrounds the adjacent pronuclei becomes concentrated at their interface. Following syngamy, the parental chromosomes line up at the equator of the zygote's first mitotic spindle



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  		FIG. 5. The contribution of different anomalies to fertilization failure after ICSI of IVM horse oocytes. These failures were categorized by comparison to the patterns observed in normally developing ICSI-fertilized horse zygotes and to abnormalities reported following ICSI of human oocytes. Basically, the causes of defective fertilization were A) failure of sperm incorporation, B) failure to complete meiotic maturation and/or defective oocyte activation, C) failure to extrude the second polar body leading to retention of both sets of maternal chromosomes within the cytoplasm and a second FPN (abnormal tripronuclear stage: Digyny), D) abnormal microtubule nucleation giving rise to a truncated or otherwise defective sperm aster, and E) inappropriate activation of the maternal spindle causing premature condensation of the male chromosomes


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study examined the cytoskeletal reorganization and chromatin configuration in horse oocytes during fertilization by intracytoplasmic sperm injection and demonstrated clearly that migration and fusion of the male and female genomes and cell cleavage during early embryonic development are accompanied by complex rearrangements of the cytoskeleton (summarized in Fig. 6). Analysis of the stage at which ICSI failed revealed that most fertilization failures were due to failure of the injected oocyte to activate (65%). Furthermore, failure of oocyte activation was often accompanied by incomplete sperm decondensation, suggesting inadequate communication between the gametes during the fertilization process. This study also demonstrated that during fertilization of horse oocytes, microtubule organization is initiated by the sperm midpiece, which, via a distinct nucleation site, orchestrates formation of the sperm aster. Thus, the zygotic centrosome in horses appears to be primarily paternally inherited, just as it is in many other mammalian species (e.g., sheep [26], cow [24], pig [25], rhesus monkey [23], rabbit [39], and humans [22]). However, microtubule assembly was also observed in 76% of sham-injected horse oocytes, mostly in the form of multiple asters dispersed randomly throughout the cytoplasm. Occasionally, these asters assembled into a more organized microtubule network, which allowed parthenogenetic development (16%). Since cell division has also been reported after parthenogenetic activation of horse oocytes [40, 41], it is concluded that the oocyte itself contains sufficient material to form a functional centrosome, just as parthenogenetic mouse zygotes are able to construct a maternal centrosome capable of duplicating and forming a functional mitotic spindle [21]. By contrast, studies on fertilization and polyspermy in cattle [24], pig [25], human [22], and rhesus monkey [23, 42] zygotes have demonstrated that the sperm aster is the most prominent, and usually the only, microtubule-containing structure in the zygote. This does not, however, rule out the possibility that the zygotic centrosome consists of both paternal and maternal components. Indeed, Simerly et al. [43] showed that in human oocytes the zygotic centrosome must be composed largely of maternally derived {gamma}-tubulin (a centrosomal protein essential for nucleation of microtubules), because the modest amount of {gamma}-tubulin present in the spermatozoa was insufficient for microtubule assembly. The recruitment of maternal {gamma}-tubulin appeared to be an important factor in the transformation of the sperm centrosome into a functional zygotic centrosome. Studies on cattle [24] and rabbit [44] zygotes and parthenotes have also supported a biparental contribution to the zygotic centrosome, and our observations on microtubule nucleation in horse oocytes after ICSI or sham injection strongly support a biparental origin of the zygotic centrosome in this species.

The variability in chronology of the cytoskeletal and nuclear rearrangements observed in horse oocytes after ICSI probably reflected both the heterogeneity of the oocyte population after collection from abattoir-derived ovaries and maturation in vitro [45] and the fact that the oocytes were not subjected to any specific activation treatment after ICSI. Despite or maybe aided by this asynchrony, the culture of injected oocytes during different periods (i.e., 6, 12, 18, 24, and 48 h) after ICSI allowed visualization of many different stages of fertilization and helped to unravel the sequence of cytoskeletal and nuclear remodeling that occurs during this process in horse zygotes. Of course, to establish the efficiency of ICSI in the horse, it is necessary to compare the events that accompany fertilization by ICSI with those occurring during fertilization in vivo. In the current study, injected oocytes began to show signs of activation, such as reorganization of the meiotic spindle and progression through meiosis II, within 6 h of sperm injection. Unfortunately, little is known about the events that occur as early as 6 h after sperm penetration in vivo, and it is not clear how long after ovulation sperm penetration occurs. Enders et al. [17] and Bézard et al. [1] were unable to detect any evidence of fertilization, e.g., sperm incorporation or oocyte activation and progression through metaphase II, in oocytes collected from the oviducts of mares earlier than 10 h after mating. However, the time required for sperm to be transported to the site of fertilization and to capacitate so that they are ready to bind to and penetrate an oocyte is unknown but could easily be longer than 6 h [46]. Nevertheless, once sperm transport is complete, it is likely that fertilization proceeds rapidly, since Torner et al. [19] noted sperm head decondensation as early as 2–4 h after the onset of coincubation of spermatozoa with IVM oocytes in an IVF system.

Large differences between oocytes were observed in the timing of male chromatin decondensation and formation of the sperm aster, which might suggest a loss of synchrony between the male and female gametes during fertilization by ICSI. The presence of the sperm acrosome and perinuclear theca on injected sperm, structures that are removed at the oolema during normal fertilization [37, 47], would presumably tend to delay sperm decondensation. With regard to the time required for horse zygotes to reach the pronuclear stage, in the current study only 50% of presumptive zygotes had reached this stage at 12 h after injection but most had done so at 18 h (Fig. 1). On the other hand, Grøndhal et al. [18] showed that pronucleus formation in vivo is completed as early as 12 h after ovulation. The apparent delay in the formation of the pronuclei after ICSI may relate to the need for greater sperm remodeling, although Torner el al. [19] reported that pronucleus formation did not peak until 16–24 h after conventional IVF. In the current study, only 10% of the presumptive zygotes had undergone cleavage 24 h after injection, but at 48 h the proportion of two-cell or later stage embryos had risen to 36% (Fig. 1). By contrast, the first cellular cleavage in vivo has been reported to occur about 20–24 h after ovulation [1, 17], with most embryos reaching the four- to six-cell stage by 48 h [1]. On the other hand, Choi et al. [14] reported that only 10% and 4% of oocytes injected with fresh and frozen-thawed spermatozoa using a Piezo drill had undergone cellular cleavage by 20 h after injection, whereas Torner et al. [19] did not detect cleavage until 32 h after the onset of conventional IVF. In conclusion, there appears to be a similar delay in the early events of fertilization and embryo development after fertilization of IVM oocytes by conventional IVF or by ICSI, and it is therefore possible that the irregularities reflect not only a delay in sperm decondensation and male pronucleus formation but also defects that arise during the process of in vitro oocyte maturation. Indeed, IVM horse oocytes are known to be developmentally compromised [48].

In the current study, a high proportion (25%) of oocytes or zygotes showed signs of fertilization failure after ICSI, and the principal reason for failure appeared to be failed oocyte activation (65%; Fig. 5). The mechanism by which oocytes activate after ICSI is itself unclear, but by analogy with other mammalian species, it must be assumed that metaphase II arrest in horse oocytes is maintained by high concentrations of metaphase-promoting factor (MPF [49]). Inactivation of MPF is one of the critical events of oocyte activation because it "unblocks" the cell cycle and allows the oocyte to complete the second meiotic division. Physiologically, MPF inactivation and oocyte activation are induced by entry of the sperm, which triggers a release of calcium from oocyte intracellular stores and sets off a series of signaling events that use calcium as a second messenger [50]. During ICSI, the injected sperm must elicit these calcium oscillations (reviewed by Tesarik [51]). However, it has been recently reported that the occurrence of sperm-induced calcium oscillations in both in vivo- and in vitro-matured horse oocytes subjected to ICSI were inconsistent [52]. Any perturbation or change in the pattern of calcium oscillation can cause incomplete MPF inactivation and thereby abnormalities of oocyte activation that are reflected in abnormal or incomplete fertilization and, in particular, a failure of the oocyte to progress through meiosis and of the sperm chromatin to properly decondense [51]. Sperm chromatin decondensation could also be negatively affected by a deficiency of those cell cycle proteins specifically required for decondensation, such as glutathione and nucleoplasmin [53, 54]. The fact that failure of sperm chromatin decondensation was observed particularly in oocytes that failed to activate, suggests a failure of the sperm to adequately transmit its activating signal to the oocyte [51] or a failure of the oocyte to respond to this signal.

Other causes of fertilization failure as described in the present study include the presence of multiple microtubule foci in the cytoplasm of injected oocytes. This defect in microtubule assembly may have been associated with incorrect reconstruction of the zygote's centrosome, as seen in arrested human IVF oocytes [55]. In this respect, defects in microtubule motor proteins, such as dynein, have been reported to result in detachment of the aster from the male pronucleus [21]. Based on the current observations, we suggest that the multiple microtubule foci originated by fragmentation of a defective meiotic spindle in oocytes that remained in meiotic arrest despite sperm incorporation. However, it cannot be ruled out that some oocytes were activated and that the sperm assisted in microtubule organization despite remaining condensed. To date, there is no information on the effect of aging of the horse oocyte on microtubule distribution. In a previous study [29], we showed that in our experimental conditions 25% of the oocytes can be expected to reach metaphase II by 24 h and an additional 17% should be in metaphase II by 36 h after IVM. This would suggest that 25% of the oocytes are aged 12 h by the time that they were selected for ICSI. However, we did not observe any obvious spindle or chromosome desegregation abnormalities in the metaphase II oocytes by 24 and 36 h of IVM that would suggest defects in their microtubule patterns. In addition, the cytoskeletal network was also presumably disrupted in those injected oocytes that displayed three pronuclei (12%). Digyny results from failure to form a second polar body and consequent retention of both sets of maternal chromosomes as pronuclei [56]. In the present study, 5% of arrested oocytes formed, in addition to the maternal meiotic spindle, an anastral paternal spindle associated with the condensed paternal chromosomes. Such premature condensation of the male chromosomes has been reported in human zygotes [57] and appears to result from failed activation of the oocyte after sperm injection and the continued presence of active chromatin condensing factors (e.g. MPF) in the ooplasm. This, in turn, prevents the transformation of the sperm nucleus into a male pronucleus and causes the sperm chromatin to condense (reviewed by Zenzes and Casper [58]).

In summary, in this study CLSM was used to reveal the way in which the cytoskeletal and nuclear events that occur during fertilization of horse oocytes by ICSI are choreographed and to demonstrate the significance of highly integrated cytoskeletal changes in the migration and fusion of the parental genomes. The comparison of the microtubular structures in zygotes and parthenotes suggests that the sperm contributes the centrosomal template during fertilization but that the oocyte contributes structural entities to the functional zygotic centrosome and to the cytoplasmic microtubule network. Failure of fertilization after ICSI was due primarily to failure of gamete activation during the very early fertilization events, and the high rate of failure observed in this study presumably relates, at least in part, to inadequacy of the in vitro-matured oocytes used. Nevertheless, until conventional IVF becomes reliable, ICSI may be the best way to produce zygotes for offspring production, to perform fundamental research into the cellular and molecular events of fertilization, and to investigate infertility and understand the cellular basis of early pregnancy failure in horses.


   ACKNOWLEDGMENTS
 
The authors would like to thank Mr. A. Klarenbeek and Mr. D Deruyck-Seghers for the supply of ovaries, Mrs. M. Bitterling-Van Weeren for her assistance with the illustrations, and Dr. T. Tharasanit for his assistance during the experiments. Confocal laser scanning microscopy was performed in the Center for Cell Imaging of the Department of Biochemistry and Cell Biology, Utrecht University.


   FOOTNOTES
 
1 Correspondence: Jordi L. Tremoleda, Department of Equine Sciences, Section Reproduction, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 12, 3584 CM Utrecht, The Netherlands. FAX: 31 30 2534811; J.Tremoleda{at}vet.uu.nl Back

Received: 29 October 2002.

First decision: 18 November 2002.

Accepted: 26 February 2003.


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 DISCUSSION
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