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Polymerization of Nonfilamentous Actin into Microfilaments Is an Important Process for Porcine Oocyte Maturation and Early Embryo Development¹

^a Department of Animal Science, University of Missouri-Columbia, Columbia, Missouri 65211

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Actin is one of the major proteins in mammalian oocytes. Most developmental events are dependent on the normal distribution of filamentous (F-) actin. Polymerization of nonfilamentous (G-) actin into F-actin is important for both meiosis and mitosis. This study examined G- and F-actin distribution in pig oocytes and embryos by immunocytochemical staining and confocal microscopy. Actin protein was quantified by electrophoresis and immunoblotting. G-Actin was distributed in the whole cytoplasm of oocytes and embryos irrespective of their stages. F-Actin was distributed at the cortex of oocytes and embryos at all stages, at the joint of blastomeres in the embryos, in the cytoplasm around the germinal vesicle (GV), and in the perinuclear area of 2- to 4-cell-stage embryos. No differences in the amount of actin protein were found among oocytes and embryos. Oocytes cultured in medium with cytochalasin D (CD), an inhibitor of microfilament polymerization, underwent GV breakdown and reached metaphase I but did not proceed to metaphase II. Two- to 4-cell-stage embryos cultured in medium with CD did not develop to blastocysts. When GV-stage oocytes or 2- to 4-cell-stage embryos treated with CD for 6 h were re-cultured in media without CD, oocytes or embryos re-assembled actin filaments and underwent a meiotic maturation or blastocyst formation similar to that of controls. These results indicate that it is the polymerization of G-actin into F-actin, not actin protein synthesis, that is important for both meiosis and mitosis in pig oocytes and embryos.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Actin has stimulated a major interest in its physical and functional significance for the study of cell biology because it plays a functional role in a number of cellular functions. Both meiosis and mitosis require the spatial and temporal coordination of cytokinesis with nuclear division. Cytoskeleton systems are important for cytokinesis in most mammalian cells [1]. Actin is one of the major proteins in mammalian oocytes from which microfilaments are polymerized. Most developmental events, such as polar body release, nuclear migration, and embryo cleavage, are dependent on normal filamentous (F-) actin distribution [2–6]. Recently, it was found that F-actin also plays an important role(s) in the distribution of some organelles, such as mitochondria [7, 8] and Golgi complexes [9], ion channel regulation [10], and the expression of some mRNAs [11]. Many of these processes require the dynamic behavior of the actin cytoskeleton, which involves polymerization and depolymerization of actin filaments [1, 12]. Most cells keep a large pool of nonfilamentous (G-) actin to maintain the ability to quickly reorganize F-actin when subjected to environmental changes [12]. More recent evidence suggests that although actin may exist as a significant physical presence within the cytoplasm, its functional concentration is actually much less, depending mainly on regulatory proteins such as profilin and ß-thymosin [13–15]. In addition, the amount of G-actin available for polymerization to F-actin is controlled by the selective binding of proteins to monomeric actin [1, 13]. Most studies on the dynamics of actin filaments have been conducted in somatic cells [1, 12–15]. In gametes, information on actin dynamics has been provided by studies in mice [2–4] and recently in pigs [5, 6]. However, the molecular studies of actin synthesis have been conducted only in Xenopus laevis [16] and mouse [17–21] eggs or preimplantation embryos. Little information has been reported on pig oocytes or embryos. This shortage of information may be due to the fewer oocytes or embryos available for such a study. Recently, a comparatively successful in vitro system for production of matured oocytes and preimplantation embryos in the pig has been established [22, 23]. This in vitro system has made it possible to produce a large quantity of oocytes or embryos for research.

In our previous studies, we found that about 30% of in vitro-matured pig oocytes developed to the blastocyst stage after in vitro fertilization [22, 23]. However, when we compared these blastocysts with those produced in vivo, a lower cell number (less than one third) was found in in vitro-produced blastocysts [23]. There were also fewer microfilaments in most pig embryos produced in vitro than in embryos produced in vivo [23], and it was suggested that fewer actin filaments in pig embryos produced in vitro was one of the reasons for low developmental ability [23]. However, it is not clear whether the amount of actin produced by pig oocytes or embryos meets the need for polymerization of F-actin under in vitro conditions, or whether inhibition of actin polymerization affects oocyte maturation and embryo development. In order to help answer these questions, in the present study we examined 1) the dynamics and distribution of F- and G-actin in pig oocytes during meiotic maturation and in pig embryos during early development, 2) actin protein content in oocytes and embryos at various stages, and 3) the effects of cytochalasin D (CD), an actin polymerization inhibitor, on microfilament distribution and the relationship of CD to oocyte maturation and embryo development.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In Vitro Maturation of Oocytes

In vitro maturation of pig oocytes was based on the procedures reported in our previous study [22]. Briefly, oocytes were aspirated from antral follicles (3–6 mm in diameter) of ovaries collected from slaughtered prepubertal gilts. After being washed 4 times with Hepes-buffered Tyrode's lactate containing 0.1% (w:v) polyvinyl alcohol (Hepes-TL-PVA), each group of 50 oocytes surrounded by compact cumulus was cultured for 44 h in tissue culture medium (TCM)-199 supplemented with 0.57 mM cysteine, 10 ng/ml epidermal growth factor (Sigma Chemical Co., St. Louis, MO), 10 IU/ml eCG, 10 IU/ml hCG, and 0.1% PVA, at 39°C in 5% CO₂ in air in a 500-µl drop of the same medium.

In Vitro Production of Embryos

After maturation, oocytes were separated from the enclosed cumulus by pipetting in maturation medium containing 0.02% hyaluronidase (Sigma). Cumulus-free oocytes were inseminated in vitro in a modified Tris-buffered medium as reported previously [22, 23]. Six hours after insemination, oocytes were removed from fertilization drops and cultured in 500 µl of culture medium (North Carolina State University [NCSU] 23 containing 4 mg/ml BSA) in a four-well culture plate until examination.

Assessments of F- and G- Actin

Oocytes or embryos used for examination of F- and G-actin were fixed by 3.7% paraformaldehyde in PBS for 2 h at room temperature. After fixation, samples were treated with 1% (v:v) Triton X-100 in PBS for 6 h at room temperature, washed twice in PBS, and cultured in PBS containing 20 mg/ml BSA and 150 mM glycine for 30 min. After being washed for another hour in PBS, samples for examination of F-actin were incubated in PBS-Tween 20 (0.1%, v:v) containing 1 µg/ml fluorescein isothiocyanate (FITC)-phalloidin (Sigma). Samples for examination of G-actin were incubated in PBS-Tween containing anti-ß-actin developed in the rabbit (Sigma; A 2066) for 1 h at 39°C. As a control, oocytes and embryos were also labeled with anti--actin (Sigma; A 2668) developed in the rabbit. After washing twice in PBS-Tween solution for 2 h at room temperature, the samples were incubated with mouse anti-rabbit IgG conjugated with FITC (Sigma; 9887) for 1 h. Nuclear status of all samples was determined by staining with 10 µg/ml propidium iodide. Finally, samples were examined using a Bio-Rad MRC-600 laser scanning confocal microscope (Bio-Rad Laboratories, Hercules, CA).

Immunoblotting and Quantification of Actin

One hundred oocytes at germinal vesicle (GV: 0 h of culture), metaphase I (M-I: 22 h of culture), and metaphase II (M-II: 44 h of culture) stages, or embryos at 2-cell (36 h after in vitro fertilization [IVF]), 4-cell (36 h after IVF), and blastocyst (6 days after IVF) stages were washed in Hepes-TL-PVA, collected in 15 µl Laemmli sample buffer (Bio-Rad), and boiled for 4 min. The proteins were separated by electrophoresis in 10% polyacrylamide gels (Bio-Rad) and electrically transferred to nitrocellulose membranes (Bio-Rad) at 4°C. The membranes were washed in 30 ml PBS containing 0.6 g BSA and 15 µl Tween-20 for 1 h at room temperature and then incubated in a solution (0.5 g dried nonfat milk, 0.002 g sodium azide, and 2 µl Tween 20 in 10 ml PBS) containing anti-ß-actin (1:70; Sigma; A 2066) for 1.5 h. A control using anti--actin was also used. After being washed 3 times for 10 min each, the membranes were further incubated in 200 ml Tris-buffered saline containing dried nonfat milk for 10 min. The membranes were finally incubated with anti-rabbit IgG conjugated with alkaline phosphate (1:200; Sigma; A 2306) for 1.5 h and developed with Sigma premixed 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) solution (Sigma; B 6404). The densities relative to M-II oocytes were calculated with a computerized Sigma Scan Imaging system (Jandel Scientific, Corte Madera, CA).

Treatment of Oocytes and Embryos with CD

Oocytes at the GV stage were cultured in maturation medium with or without (control) 5 µM CD for 44 h. Subgroups of oocytes were cultured for 6 h in medium with CD and then cultured in medium without CD until 44 h. After culture, cumulus cells were removed completely, and oocytes were fixed for examination of nuclear status and actin filament distribution. Embryos at 2- to 4-cell stages collected at 36 h after IVF were cultured in medium containing 5 µM CD for 6, 12, 40, and 108 h, and then fixed for examination of microfilament and nuclear status. In order to examine whether CD-treated embryos developed further, embryos cultured for 6 h in medium with CD were washed completely and then re-cultured in medium without CD until 6 days after IVF. Some embryos were also cultured in medium without CD. After completion of culture, blastocyst formation in each group and microfilament distribution in the embryos were examined by the methods described above.

Statistical Analysis

Four replicate trials were conducted, and all percentage data were subjected to arc sine transformation before statistical analysis. The transformed data and the average cell numbers in blastocysts were compared by ANOVA. A value of P < 0.05 was considered to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Distribution of F- and G-actin in Oocytes and Embryos

Oocytes were cultured for 0, 22, or 44 h before fixation. Two- to 4-cell-stage embryos were obtained 36 h after IVF, and blastocysts were obtained 6 days after IVF. As shown in Figure 1, A–F, when anti-ß-actin was used, it was found that G-actin was distributed in the whole cytoplasm of oocytes or embryos irrespective of their stages. However, no positive staining was observed when anti--actin was used. Some differences were observed in F-actin distribution in oocytes and embryos. In GV-stage oocytes, F-actin was distributed at the cortex and cytoplasm around the GV (Fig. 1G); in M-I oocytes, F-actin was distributed at the cortex, with a thicker area near the chromosomes (Fig. 1H); in M-II oocytes, F-actin was distributed at the cortex of the oocyte and partial cytoplasm (Fig. 1I); in 2- to 4-cell-stage embryos, F-actin was distributed at the cortex of blastomeres (thicker at the joint of cells than in other areas of the cortex) and perinuclear cytoplasm in all or part of the blastomeres (Fig. 1, J and K); and in blastocysts, F-actin was distributed at the cortex of blastomeres and formed a filamentous net (Fig. 1L).

View larger version (47K): [in this window] [in a new window]   FIG. 1. Distribution of G-actin and F-actin in pig oocytes and embryos. G-Actin (A–F) was stained by FITC-conjugated mouse anti-rabbit IgG after labeling with anti-ß-actin, while F-actin was stained by FITC-phalloidin (G–L). The associated nuclei were stained with propidium iodide and are shown on the right side of the oocytes or embryos (A'–L'). Oocytes were stained at the GV (AA' and GG'), M-I (BB' and HH'), and M-II (CC' and II') stages, and the embryos were at 2-cell (DD' and JJ'), 4-cell (EE' and KK'), and blastocyst (FF' and LL') stages. G-Actin was distributed in the cytoplasm of oocytes or embryos irrespective of their stages (A–F), and F-actin was distributed in the cortex of oocytes (G–I) and embryos (J–L), and also in cytoplasm around the nucleus (G, J, and K). A thick area was also observed in the M-I oocyte close to the chromosome, which was related to the release of the first polar body (arrow in H).

Quantification of Actin in Oocytes and Embryos

When anti--actin was used to examine the actin protein, no band was observed in any sample. However, when anti-ß-actin was used, one band was observed in all samples, but no differences were observed in the quantity of actin protein from oocytes (GV to M-II) and embryos (2-cell stage to blastocyst stage). The relative density of M-II oocytes was 118.1 ± 37.7% for GV-stage oocytes, 105.9 ± 28.4% for M-I-stage oocytes, 104.6 ± 14.6% for 2-cell-stage embryos, 109.7 ± 15.0% for 4-cell-stage embryos, and 91.3 ± 29.4% for blastocysts (Fig. 2).

View larger version (73K): [in this window] [in a new window]   FIG. 2. Quantification of ß-actin in pig oocytes and embryos. The top panel shows the result from one trial of Western blotting of the samples from one hundred oocytes or embryos. The lower panel shows the relative density to M-II oocytes from four replicate trials and no differences were observed. blast, Blastocysts

Effects of CD on Actin Filament Distribution and Morphology of Oocytes and Embryos

When oocytes at the GV stage were cultured for 44 h in medium with CD, as shown in Table 1, most oocytes (87%) underwent GV breakdown and reached M-I, but very few (1%) reached M-II. However, when the oocytes were treated for 6 h in medium with CD and then re-cultured in medium without CD, the same proportion (82%) of oocytes proceeded to M-II, and no difference from controls was observed (82%). When oocytes were cultured in medium with CD, as shown in Figure 3, no filaments were present in the oocytes (Fig. 3, A'–D'), the oocytes were at GV or M-I stages (Fig. 3, A and B), or the chromosomes were separated in the cytoplasm but the first polar body was not released from the oocytes (Fig. 3, C and D).

View larger version (14K): [in this window] [in a new window]   FIG. 3. Micrographs of pig oocytes that have been cultured in medium with CD for 44 h from beginning of culture. A–D) Nuclear status is shown. Arrows in C and D show separating and separated chromosomes, respectively. A'–D') FITC-phalloidin staining of the same oocytes as in A–D. No microfilaments were observed in these oocytes

When embryos at 2- to 4-cell stages were treated with CD for 6 h, as shown in Table 2, microfilaments at the cortex began to disappear, and more microfilament foci were formed around the perinuclear cytoplasm (Fig. 4, A' and B'). When the embryos were treated for more than 12 h, all of the treated embryos were without microfilaments at either cortex or joints of blastomeres, and most (84–100%) embryos did not have microfilaments in the perinuclear area (Fig. 4, C' and D'), although some had normal morphology (one nucleus per blastomere as shown in Fig. 4, A–C). As shown in Figure 5, when CD treatment time was increased, the embryos with binucleate blastomeres (two nuclei per blastomere in at least one blastomere of the embryo, as shown in Fig. 4D) increased from 9% of controls to 23%, 59%, 79%, and 85% of the embryos treated for 6 h, 12 h, 40 h, and 4.5 days. When 2- to 4-cell-stage embryos treated for 6 h with CD were re-cultured in medium without CD for 4.5 days (6 days after IVF), 56.6% developed to blastocysts, a blastocyst rate similar to that in controls (61.6%). No differences were observed in the mean cell number per blastocyst between CD-treated and control embryos. However, no embryos cultured in medium with CD during the total culture period developed to blastocysts (Table 3).

View larger version (20K): [in this window] [in a new window]   FIG. 4. Micrographs of pig embryos that have been cultured in medium with CD. AA') A 2-cell-stage embryo that has been cultured in medium with CD for 6 h shows one nucleus per blastomere (A), but microfilaments were destroyed at the cortex and joint of the blastomere (A'). BB') A 4-cell-stage embryo that has been cultured in medium with CD for 6 h shows one nucleus per blastomere (B), and most microfilaments in the cortex and joint of blastomeres have been destroyed (B'). CC') One 4-cell-stage embryo that has been cultured in medium with CD for 12 h shows normal nuclear status (C), but no microfilaments were detected in the embryo (C'). DD') One 4-cell embryo that has been cultured in medium with CD for 40 h shows binucleate formation in all of the blastomeres (D), and no microfilaments were observed in the embryo

View larger version (34K): [in this window] [in a new window]   FIG. 5. Effects of CD on the morphology of pig embryos cultured in vitro. Four types of morphologies were observed: normal (one nuclear per blastomere), fragmentation (one or more than one blastomere did not have nuclei), binucleate (one or more than one blastomere had two nuclei), and both fragmentation and binucleate embryos. As culture time increased in medium with CD, embryos with binucleate morphology increased significantly, and most embryos showed binucleate morphology when they were cultured for more than 40 h in medium with CD. abcdP < 0.05

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Actin is a major component of the cytoskeleton—the fibrous array that pervades almost every kind of cell including gametes and gives it a special characteristic. During oocyte maturation, fertilization, and embryo development, polymerization of actin filaments is a very important process. Early studies on the dynamics of actin filaments have been focused only on oocyte maturation and activation [2–6]. However, our previous study in the pig indicated that in vitro-produced embryos had less F-actin in their cytoplasm than in vivo-produced embryos [23]. In the present study, we extended our previous results and report dynamic behavior of G- and F-actin distribution in both oocytes and embryos. We indicate here that in vitro-produced pig oocytes and embryos at the stages examined have the same amount of, and possibly sufficient, G-actin in the cytoplasm but most G-actin is not polymerized to its filamentous status, F-actin, especially in embryos. Such results suggest that the conditions used in the present study for embryo development are not optimal for actin polymerization. Although the conditions that can accelerate actin polymerization are not reported here, some modification in culture conditions that will satisfy such an important intracellular change seems necessary. Studies in the hamster [7] and rat [24] have indicated that phosphate in culture medium was an inhibitor for such a process, but phosphate at a concentration of 1.19 mM did not block pig embryo development in culture when the embryos were collected from the oviducts [25]. Whether phosphate also affects the actin polymerization in pig embryos or whether phosphate at a higher concentration inhibits pig embryo development remains to be clarified. It is possible that phosphate (1.19 mM) used in the present study partially inhibits both actin polymerization and embryo development.

How G-actin is polymerized into F-actin in zygotes is not clear. Studies in somatic cells indicate that intracellular ion changes and some actin-binding proteins are important regulators for actin assembly and disassembly [14, 15]. One of them is intracellular calcium change. These results may also be possible in oocytes, as intracellular calcium release during oocyte activation plays a central role in initiation of embryo development, and one of the events associated with cleavage is actin filament formation after oocyte activation. The distribution of actin filaments in the oocytes before and after activation was different, with more cytoplasmic filaments, especially peri-pronuclear actin filaments, formed after oocyte activation [6]. Perinuclear actin filaments were also observed in early pig embryos collected from oviducts [23]. However, when the embryos produced by in vitro maturation/fertilization/development entered into the 2-cell stage or beyond, we found that cytoplasmic F-actin significantly decreased and was detected only in some blastomeres of the embryos [23]. Also, the proportion of embryos with cytoplasmic F-actin in all blastomeres decreased as development proceeded. These results suggest that polymerization of G-actin to F-actin was affected by some factor(s) during culture that remains to be clarified. One possibility is that the culture conditions do not completely meet the physiological criteria for embryo growth in vitro. This may also be the reason that in vitro-produced embryos develop more slowly and have lower cell numbers than in vivo-produced blastocysts [23].

The finding that actin filaments are important for oocyte maturation and embryo development is also supported by the results obtained by adding CD to inhibit polymerization of actin filaments during culture. Abundant cytoplasmic microfilaments were found in oocytes at the GV stage but were reduced after oocytes underwent GV breakdown and reached M-I. This result may suggest the false conclusion that microfilaments are necessary for GV breakdown. However, when GV-stage oocytes were cultured in medium containing CD, GV breakdown and M-I progress were not inhibited. By contrast, the transition from M-I to M-II was prevented. These results clearly indicate that microfilaments do not participate in GV breakdown but do participate in polar body release, which is consistent with previous studies reported in the mouse [3, 4]. Furthermore, most oocytes cultured in medium with CD showed an M-I metaphase plate, and only a few oocytes showed separated chromosomes. Therefore, it seems that microfilaments are also related to normal function of microtubules, as microtubules are the main regulator for separation of chromosomes [3]. The presence of separated chromosomes in some oocytes may have been due to separation before microfilaments and/or microtubules were completely depolymerized.

Binucleate blastomeres and fragmentation of pig embryos produced in vitro are serious problems, which may result in lowered developmental rate and reduced cell number of the blastocysts [23]. There are possibly many reasons for these abnormalities, which are poorly understood. Abnormal actin filament distribution is suggested to be one of the reasons. When embryos were treated with CD, we found that embryos with binucleate blastomeres increased in a time-dependent manner. Observations in this study clearly indicate that the formation of binucleate cells resulted from failure of polymerization of microfilament in the embryos. In addition, most embryos showed only binucleate cells when cultured in the medium with CD. These results indicate that short-term CD treatment mainly affected cytokinesis, but long-term treatment also affected the karyokinesis, as most embryos showed binucleate blastomeres when the culture time was prolonged to 4.5 days. It is also possible that extended treatments were actually toxic to the nucleus and caused apoptosis, inhibited protein synthesis [26], or altered the impedance of membrane [27].

We did not find -actin in the oocytes and embryos by using immunocytochemically staining and immunoblotting. However, ß-actin was present in all samples examined. Our results were the same as those observed in the Xenopus laevis, in which it was found that -actin is detectable only in late gastrulae [16]. Results in the present study indicate that only ß-actin is present in pig oocytes and embryos at the early stages studied. ß-Actin was at the same level at all stages, from oocytes at the GV stage to embryos as blastocysts. A previous study in the mouse indicated that actin was synthesized during oocyte growth and stopped in the grown oocytes [17]. Whether in vitro culture affects actin synthesis is not clear. However, it seems that pig oocytes or embryos do have sufficient actin protein, but most actin is in a nonfilamentous state and is not sufficiently polymerized to microfilaments under these culture conditions. This conclusion was also supported by our other study, in which we found that when TCM 199 was used to culture pig embryos, no embryos (2–4-cell stages) developed to the blastocyst stage and no microfilaments were found in the cytoplasm of the blocked embryos (unpublished results).

In conclusion, the results obtained in the present study indicate that most actin in pig oocytes and embryos is present in a nonfilamentous state. Polymerization of G-actin to F-actin may be of importance for pig oocyte maturation and embryo development. Inhibition of actin filament polymerization prevents completion of oocyte meiosis and embryo development.

	ACKNOWLEDGMENTS

 
We thank B. Nichols for secretarial assistance in preparation of the manuscript, and T. Cantley and R. Cabot for transport of samples.

	FOOTNOTES

 
First decision: 17 June 1999.

¹ This research is supported in part by the National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Development, funded by the National Institutes of Child Health and Human Development, NIH, through cooperative agreement HD34588. This manuscript is a contribution from the Missouri Agricultural Experiment Station, Journal Series Number 12,896.

² Correspondence: Billy N. Day, 159 Animal Sciences Research Center, Department of Animal Science, University of Missouri-Columbia, Columbia, MO 65211. FAX: 573 884 7827; dayb{at}missouri.edu

³ Current address: Division of Reproductive Medicine and Infertility, Women & Infants' Hospital of Rhode Island, Brown University School of Medicine, Providence, RI 02905.

Accepted: December 9, 1999.

Received: May 17, 1999.

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