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Biology of Reproduction 65, 1579-1585 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Development of In Vitro-Matured Oocytes from Porcine Preantral Follicles Following Intracytoplasmic Sperm Injection

Ji Wu1,a, Douglas T. Carrell, and Aaron L. Wilcoxa

a Division of Urology, University of Utah School of Medicine, Salt Lake City, Utah 84132

ABSTRACT

The objective of this study was to assess fertilization and embryonic development following intracytoplasmic sperm injection (ICSI) of oocytes from porcine preantral follicles matured in vitro. Also, another aim was to describe actin filament distribution during fertilization and embryonic development of those oocytes after ICSI as one of the factors assessed. Preantral follicles isolated from prepubertal porcine ovaries were cultured in a system that supports follicular development. After in vitro maturation, the oocytes were fertilized by ICSI or conventional fertilization in vitro (IVF). Actin filaments of the fertilized oocytes and embryos produced by ICSI or IVF were stained by rhodamine-phalloidin and visualized by fluorescence microscopy. ICSI resulted in 64% fertilization of porcine preantral follicle oocytes matured in vitro. Of those, 51% of the fertilized oocytes cleaved and 21% developed to the blastocyst stage. No significant differences in percentages of oocyte fertilization, cleavage, and blastocyst formation were observed between ICSI and IVF (53%, 45% and 16%, respectively). Actin filament distribution during fertilization and embryonic development of ICSI- or IVF-fertilized oocytes from porcine preantral follicles was similar to that of oocytes derived from antral follicles and fertilized by standard IVF. These results indicate that oocytes from porcine preantral follicles matured in vitro following ICSI can undergo fertilization and subsequent embryonic development.

assisted reproductive technology, fertilization, follicle, gamete biology, oocyte development

INTRODUCTION

Hiramoto [1] was the first to record embryological development following injection of sperm cells into the eggs of sea urchins in 1962. The first intracytoplasmic sperm injection (ICSI) of mammalian eggs was conducted by Uehara and Yanagimachi in 1976 [2]. The development of ICSI has been the most significant assisted reproductive advancement in the 1990s. ICSI of oocytes from in vivo- or in vitro-matured antral follicles has been used to generate live human [3], rabbit [4], sheep [5], bovine [6], equine [7], and murine offspring [8]. In swine, Catt and Rhodes [9] were the first to report attempts to inject porcine spermatozoa into oocytes from antral follicles and to verify the presence of pronuclei and sperm tails in the vitellus. In two recent studies, ICSI of porcine oocytes from antral follicles using immature round spermatids yielded embryos that were capable of blastocyst development in vitro [10]. Porcine embryos derived from in vivo-matured oocytes fertilized by ICSI can develop into live pigs [11]. However, post-ICSI development of oocytes from in vitro-matured porcine preantral follicles has not been reported. This may be due to the greater follicle dimension in large animals and to the presence of a thick theca that restricts the transport of nutrients and gases during the long-term culture period required for follicle culture. We [12] have developed an in vitro culture system capable of supporting porcine follicle growth from the preantral to antral stages, oocyte maturation, fertilization, and embryonic development. This culture system offers the possibility of embryonic development of in vitro-matured preantral follicle oocytes following ICSI.

Actin is a major component of the cytoskeleton. It has been suggested that actin filaments are related to embryonic normality [13]. Many developmental events in oocytes and embryos, such as polar body formation, nuclear migration, and mitotic cleavage are dependent on normal distribution of actin filaments [14–20]. Kim et al. [20] have examined actin filament distribution during fertilization and parthenogenesis in porcine oocytes in antral follicles. Recently, Wang et al. [13] demonstrated the existence of a relationship between actin filament distribution and embryo development in porcine embryos from antral follicle oocytes. However, actin filament distribution during fertilization and embryonic development of porcine preantral follicle oocytes following ICSI remains unknown.

The objective of this study was to assess 1) fertilization and embryonic development of oocytes from porcine preantral follicles matured in vitro following ICSI and 2) actin filament distribution during fertilization and embryonic development of those oocytes after ICSI.

MATERIALS AND METHODS

Animal and Tissue Collection

Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory in Dulbecco PBS (DPBS; Gibco 11500-030, Grand Island, NY) supplemented with 3 mg/ml BSA (A 8022, fraction V; Sigma, St. Louis, MO) maintained at 30–37°C. Blood from prepubertal gilts was collected and centrifuged at 300 x g for 10 min. The serum was removed from the pellet of cells and subsequently stored at -20°C until use.

Preantral Follicle Collection and Culture

The preantral follicles were collected and cultured using a minor modification of a protocol described by Wu et al. [12]. The ovaries were cut into small pieces (1–3 mm) and preantral follicles were isolated by dissection in DPBS with 3 mg/ml BSA. Preantral follicles with a diameter of 296 ± 9 µm (Fig. 1) were collected into 4-well multidishes (Nunclon, Nunc, IL) containing collecting medium. This medium consisted of NCSU 23 (Sigma) supplemented with 3 mg/ml BSA (A 8022, fraction V; Sigma). After collection, the follicles were transferred from the collecting medium into a culture medium, which was NCSU 23 supplemented with 3.5 µg/ml insulin (I 5523; Sigma), 10 µg/ml transferrin (T5391; Sigma), 100 µg/ml L-ascorbic acid (A 4544; Sigma), 7.5% porcine serum, and 1.5 ng/ml ovine FSH (oFSH-20, 4453 IU/mg; the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK], Torrance, CA). The follicles were cultured for 3 days in 24-well cell culture clusters plates (3524; Costar, Corning, NY) with three follicles per well in 280 µl of culture medium. The culture was carried out at 38.5°C in an atmosphere of 5% CO2 in air. The diameters of follicles (including the theca layer) were measured using a stereomicroscope ocular scale at a magnification of 50x. The culture medium was changed every day with freshly prepared culture medium.



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  		FIG. 1. Examples of living follicles at start size and ICSI. A) Preantral follicle on Day 0 of culture. B) ICSI of mature oocyte from porcine preantral follicle. Bar = 25 µm

In Vitro Maturation of Oocyte-Cumulus Complexes

Maturation of oocyte-cumulus complexes (OCCs) was performed using a modification of the procedures described by Wu et al. [12] and Abeydeera et al. [21]. Briefly, at the end of culture, the follicles were opened using two omnican needles and OCCs were flushed into DPBS supplemented with 3 mg/ml BSA. After washing three times in NCSU 23 medium supplemented with 0.23 mM pyruvate and 10% porcine serum, the OCCs were transferred to the same medium, which was further supplemented with 0.12 µg/ml oFSH, 2.5 µg/ml ovine LH (oLH-26; the National Hormone and Pituitary Program of NIDDK), 20 ng/ml epidermal growth factor (EGF; E 4127; Sigma), and 50 µg/ml L-ascorbic acid. The OCCs were cultured for 48 h with 10–12 follicular shell pieces (FSPs), containing both granulosa and theca cells from antral follicles.

In Vitro Fertilization and ICSI

After in vitro maturation, mature oocytes (the presence of the first polar body) were fertilized by conventional in vitro fertilization (IVF; control) or ICSI. IVF and ICSI were performed using the method described previously [11, 12, 22]. In brief, these oocytes were washed three times with IVF medium consisting of modified Tris-buffered medium, 2 mM caffeine, and 2 mg/ml BSA (A7888; Sigma). The oocytes were then transferred to 50 µl of IVF medium, and incubated in the incubator for about 30 min until spermatozoa were added for fertilization. Porcine sperm (SGI, Cambridge, IA) preparation was the same as that described previously [12, 23]. After sperm preparation, 50 µl of the sperm suspension was added to 50 µl of the medium that contained oocytes (final concentration of 5 x 105 cells/ml). The oocytes were incubated with spermatozoa for 5–6 h at 38.5°C in an atmosphere of 5% CO2 in air. ICSI was conducted with aid of a pair of Narishige micromanipulators mounted on a Nikon (Tokyo, Japan) inverted microscope. Porcine semen (1 ml; SGI) was washed, and the pellet was resuspended in 10 ml of DPBS supplemented 3 mg/ml BSA (A8022; Sigma). Ten 4-µl drops of flushing medium (human tubal fluid with Hepes buffer) with 10% porcine serum were arranged in two rows on the lid of a 50- x 9-mm Petri dish. The third row consisted of a 20-µl drop of sperm suspension and a 20-µl drop of polyvinylpyrrolidone (PVP) solution. All drops were covered with mineral oil (0003-0559-52; Squibb, Princeton, NJ) equilibrated overnight to 5% CO2. Each drop of flushing medium was occupied by a single oocyte whose cumulus cells were removed in 80 IU/ml hyaluronidase (SAGE Biopharma, Bedminster, NJ). Motile spermatozoa were transferred from the drop of sperm suspension into the PVP drop with an injection pipette. The sperm were immobilized by vigorous swiping of the tail. A single sperm was then aspirated into the injection pipette tail-first. The oocyte was captured by the holding pipette and immobilized with its polar body at the 7 o'clock or the 11 o'clock position [24] (Fig. 1B). The oocyte was penetrated with the injection pipette containing the sperm, and a small amount of cytoplasm was aspirated. The sperm was then injected into the center of the oocyte.

Embryo Culture and Morphologic Evaluation

After the oocytes were fertilized by IVF or ICSI, they were cultured in 500 µl of embryo culture medium (NCSU 23 containing 3 mg/ml BSA and 0.5% [v/v] MEM amino acids solution [Gibco 11130-051]) in a 60- x 15-mm center-well organ culture dish (Becton Dickinson, Falcon, Lincoln Park, NJ) for 48–168 h. Embryos at different stages of development were separately mounted on slides and fixed for 48–72 h in 25% (v/v) acetic acid in ethanol, stained with 1% (w/v) orcein in 45% (v/v) acetic acid, and examined under a phase-contrast microscope at a magnification of 200x and 400x. Embryo morphology was evaluated for 1) morphological normality (embryos with each blastomere having one nucleus), 2) fragmentation (embryos with one or more blastomeres having no nuclei), and 3) others (including embryos with one or some blastomeres having two nuclei and embryos with both fragmented and binucleate blastomeres).

Actin Filament Visualization

Actin filament distribution was assessed using the method described previously [13]. In brief, after fixation, treatment with 1% (v/v) Triton X-100 in PBS and then with a blocking solution (PBS containing 2% BSA and 150 mM glycine), the embryos were stained with 10 IU/ml rhodamine-phalloidin (Molecular Probes, Eugene, OR) for 1 h at 39°C in PBS-Tween 20 (0.1%, v/v). After washing, the embryos were stained with 100 nM Yo-Pro-1 iodide (Molecular Probes) for 5–10 min. Finally, the embryos were mounted on slides and kept in the dark (24–96 h) until viewing by fluorescence microscopy.

Statistical Analysis

Experiments were repeated at least four times. Chi-square test and one-way ANOVA were used for statistical comparisons. P values of < 0.05 were considered significant.

RESULTS

Fertilization and Embryonic Development of Oocytes from Porcine Preantral Follicles Following ICSI

The distributions of follicle sizes at the start between the ICSI and IVF (control) groups were not significantly different. Table 1 shows that ICSI resulted in 64% fertilization of porcine preantral follicle oocytes matured in vitro; 51% of the fertilized oocytes cleaved, and 21% developed to the blastocyst stage. No significant differences in percentages of oocyte fertilization, cleavage, and blastocyst formation were observed between ICSI and IVF (53%, 45%, and 16%, respectively; P > 0.05).


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  		TABLE 1. Fertilization and embryonic development of oocytes from preantral follicles following ICSI and IVF

Morphology of Porcine Embryos from Preantral Follicle Oocytes after ICSI

As shown in Figure 2, there were no marked differences in the morphology of embryos produced through ICSI and IVF. In ICSI- or IVF-derived two- to four-cell embryos, clear blastomeres were resolved. A perivitelline space (PVS) was observed in both ICSI and IVF two-cell embryos. At the four-cell stage, the PVS was filled with blastomeres. Expanded blastocysts were seen in both groups. The blastocysts derived from ICSI and IVF contained an average of 31.3 ± 2.3 and 29.8 ± 2.9 nuclei, respectively, when fixed and stained with orcein. There were no significant differences.



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  		FIG. 2. Morphology of porcine embryos from preantral follicle oocytes produced in ICSI (A, C, and E) and IVF (B, D, and F). There were no marked differences in the morphology of embryos produced through ICSI and IVF. Bar = 25 µm

The nucleus-blastomere relationship at the two- to eight-cell stages is summarized in Figure 3. Similar percentages of normal embryos and fragmentation were observed in ICSI (69% and 14%, respectively [n = 86]) and IVF (67% and 13%, respectively [n = 88]) two-cell embryos (Fig. 3A). No significant differences in the percentages of normal three- to four-cell embryos and fragmentation were seen between the two groups: 41% (n = 69) and 39% (n = 70) for normal embryos, and 32% and 30% for fragmentation in ICSI and IVF three- to four-cell embryos, respectively (Fig. 3B). Again, similar incidences of normal embryos and fragmentation were found in ICSI (29% and 50%, respectively [n = 58]) and IVF (29% and 48%, respectively [n = 56]) five- to eight-cell embryos (Fig. 3C).



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  		FIG. 3. The nucleus-blastomere relationship at two- to eight-cell stage embryos from preantral follicle oocytes produced in ICSI and IVF. A) Two-cell stage. B) Three- to four-cell stage. C) Five- to eight-cell stage

Actin Filament Distribution During Fertilization and Embryonic Development of Porcine Preantral Follicle Oocytes Following ICSI

During pronuclear formation, actin filaments were concentrated in the female nucleus and sperm head after ICSI or IVF (Fig. 4A). At the time of pronuclear apposition, actin filaments became concentrated to both the male and female pronuclei (Fig. 4B). During the embryonic stage, actin filaments were present in the cortex and at the junctions of blastomeres of embryos. Considerable differences existed in the perinuclear actin filament distribution of embryos. Twenty-eight percent (17 of 62) of two- to four-cell embryos had perinuclear actin filaments (Fig. 4C). Some embryos (19% [12 of 62] of two- to four-cell) did not have perinuclear actin filaments in their blastomeres. Most embryos (53% [33 of 62]) had partial perinuclear actin filaments in their blastomeres, including partial binucleate (asynchronized cytoplasmic and nuclear division) perinuclear actin filaments (Fig. 4, D and E). There were no significant differences in percentages of two- to four-cell embryos with perinuclear actin filaments or partial perinuclear actin filaments or without perinuclear actin filaments between ICSI and IVF (23% [11 of 48], 60% [29 of 48], and 17% [8 of 48], respectively). Figure 4F shows an example of actin filament distribution of blastocysts.



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  		FIG. 4. Actin filament distribution during fertilization and embryonic development of porcine preantral follicle oocytes following ICSI. Actin filaments were distributed in the cortex and at the junctions of blastomeres. A) Actin filaments were concentrated to the female nucleus ({triangledown}) and sperm head ({Downarrow}) at the stage of pronuclear formation. Original magnification x200. B) Actin filaments were concentrated in both the male ({Downarrow}) and female ({triangledown}) nuclear structure during pronuclear movements. Original magnification x600. C) Normal morphology and actin filament distribution (perinuclear actin filaments) were observed in the two-cell embryo. Original magnification x600. D) Abnormal actin filament distribution (partial or without perinuclear actin filaments in its blastomeres) were seen in the embryo. Original magnification x400. E) The embryo has both abnormal morphology and abnormal actin filament distribution (partial binucleate or without perinuclear actin filaments in its blastomeres). Original magnification x400. F) An example of actin filament distribution of blastocysts. Original magnification x200

DISCUSSION

The results presented in this study indicate that oocytes from porcine preantral follicles matured in vitro following ICSI can undergo fertilization and subsequent embryonic development. The ability to obtain mature, viable oocytes following culture of preantral follicles is significant because these oocytes could provide a valuable resource for use in various production systems. This study demonstrates that apparently normal embryos can be produced following IVF or ICSI of in vitro-matured oocytes derived from preantral follicles. It would be possible to use these oocytes for other purposes, such as transgenics.

This study showed that there were no significant differences in the percentages of oocyte fertilization, cleavage, and blastocyst formation when comparisons were made between ICSI and conventional IVF. The present results also indicate that the mean number of nuclei observed in IVF-derived blastocysts was similar to the number of nuclei observed in ICSI-derived blastocysts. These results support the following conclusions: 1) that the complex set of interactions normally required for sperm to penetrate the egg investments and to fertilize the egg were biologically unnecessary in mammals [25, 26], and 2) that ICSI was not detrimental to embryonic development [27–29]. These findings are consistent with previous studies [30–32]. However, these results differ from those of Griffiths et al. [33] and Shoukir et al. [34] in which the proportion of embryos forming blastocysts was smaller after ICSI than after conventional IVF. The difference between the present results and those reported by Griffiths et al. [33] and Shoukir et al. [34] may be due to differences in species, source of oocytes, the ICSI procedure itself, or paternal influence and other factors.

When spermatozoa are injected directly into oocytes, the sperm acrosome and membrane may remain intact, fusion of oocyte and sperm membranes does not occur, and a variety of Ca2+ responses occur (in human, [35, 36]). After ICSI, half the oocytes did not have oscillations in Ca2+ concentrations but underwent a prolonged Ca2+ increase. The remainder had Ca2+ oscillations but without the initial series of rapid transients. In a similar study of mouse oocytes, however, no marked differences were found between the pattern of oscillations after ICSI and conventional IVF, except for a delay in the onset of Ca2+ oscillations [37]. By contrast, during conventional IVF the fertilizing spermatozoon loses its acrosome while penetrating the zona pellucida. The sperm membrane then fuses with the oolemma and triggers a series of transient increases in cytoplasmic Ca2+ that initiate the cortical reaction and the completion of meiosis [38]. The work by Ahmadi and Ng [31] suggests no negative effect on embryonic development due to the presence of intact sperm membranes from ICSI. However, differences in the membrane structure of zygotes arising from IVF and ICSI may have physiological significance. For instance, zygotes obtained by conventional IVF have a mosaic membrane structure that results from the incorporation of the fusing sperm membrane and the cortical granules with the oolemma [39, 40]. The incorporation of the sperm membrane into the oolemma was found to have a role in the inhibition of polyspermy that was independent of cortical granule release [41]. Our finding of no difference in the morphology of the embryos between IVF and ICSI is in agreement with those of Ahmadi and Ng [31].

Kim et al. [20] demonstrated that two domains (thick and thin actin filament domains) are found in porcine mature oocyte cortex. The thick actin filament domain of the cortex may be important for polar body extrusion and normal development during fertilization because chromosomes are located in this domain. In the present study, actin filaments in porcine oocytes from preantral follicles concentrated in both the male and female chromatin after ICSI or IVF. This is consistent with the observation of porcine antral follicle oocytes after IVF by Kim et al. [20]. The actin filaments above both nuclear structures may arise from a polymerization of actin recruited from the oocyte cytoplasm.

The polymerization and depolymerization of actin filaments is an important process during oocyte maturation, fertilization, and embryo development. Abnormal actin filament distribution results in abnormal cell function [42, 43]. The present study showed 1) that actin filaments were present at the cortex and at the junctions of blastomeres of embryos from preantral follicle oocytes after ICSI or IVF and 2) that one-third of two- to four-cell embryos had perinuclear actin filaments, and that two-thirds of these embryos had abnormal actin filament distribution. These results are similar to previous observations with porcine embryos from antral follicle oocytes after IVF, but different from those with porcine embryos produced in vivo [13]. In abnormal actin filament distribution of embryos, the presence of binucleate cells may be due to abnormal polymerization of actin filaments, which results in nuclear division without cytoplasmic division. Asynchronized nuclear and cytoplasmic division indicates that culture conditions may delay the progression of polymerization and depolymerization of actin filaments [13]. Embryo fragmentation may be associated with an absence of perinuclear actin filaments in some blastomeres.

In conclusion, this study is the first to report that oocytes from in vitro-matured porcine preantral follicles following ICSI can undergo fertilization and subsequent embryonic development. There is no difference in morphology of ICSI-derived and IVF-derived embryos from porcine preantral follicle oocytes. Actin filament distribution during fertilization and embryonic development of porcine preantral follicle oocytes after ICSI or IVF is similar to that of porcine antral follicle oocytes after IVF.

ACKNOWLEDGMENTS

The authors thank the National Hormone and Pituitary Program, NIDDK, for its generous donation of FSH and LH.

FOOTNOTES

First decision: 20 March 2001.

1 Correspondence: Ji Wu, Room 302, 1247 W. Westmoreland St., Philadelphia, PA 19140. FAX: 215 707 2966; jiwu_99{at}yahoo.com Back

Accepted: July 10, 2001.

Received: February 13, 2001.

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