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Regular Article |
a Genetic Diversity
b Developmental Biology Departments, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan
c Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229-8501, Japan
d Prime Tech Ltd., Tsuchiura, Ibaraki 300-0841, Japan
e Department of Research Planning and Coordination, National Institute of Livestock and Grassland Science, Kukizaki, Ibaraki 305-0901, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, in vitro fertilization, oviduct
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Porcine ovaries were obtained from prepubertal cross-bred gilts (Landrace x Large White) at a local slaughterhouse and transported to the laboratory at 35°C. Cumulus-oocyte complexes (COCs) were collected from follicles 3–5 mm in diameter in Medium 199 (with Hanks salts; Gibco, Life Technologies Inc., Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (Gibco), 20 mM Hepes (Dojindo Laboratories, Kumamoto, Japan), 100 U/ml penicillin G potassium (Sigma Chemical Co., St. Louis, MO), and 0.1 mg/ml streptomycin sulfate (Sigma) [14]. IVM of oocytes was carried out as reported previously [12]. In brief, about 50 COCs were cultured in each 500 µl of maturation medium, a modified North Carolina State University (NCSU)-37 solution [15] containing 10% (v/v) porcine follicular fluid, 0.6 mM cysteine, 1 mM dibutyryl cAMP (dbcAMP; Sigma), 10 IU/ml eCG (PMS 1000 Tani NZ; Nihon Zenyaku Kogyo, Koriyama, Japan), and 10 IU/ml hCG (Puberogen, 500 U; Sankyo, Tokyo, Japan) in four-well dishes (Nunclon Multidishes; Nalge Nunc International, Denmark) for 20–22 h. They were subsequently cultured in the maturation medium without dbcAMP and hormones for 24 h. The maturation culture was carried out under 5% CO2 in air (20% O2) or under conditions of O2, CO2, and N2 adjusted to 5%, 5%, and 90% (5% O2), respectively, at 39°C.
IVF and IVC of Porcine Oocytes
Epididymides from a boar of the Landrace breed were collected, and epididymal spermatozoa were collected and frozen [16]. Spermatozoa were thawed and preincubated for 1 h at 37°C in Medium 199 adjusted to pH 7.8 [17]. Fertilization medium for porcine oocytes (Pig-FM) [18] consisting of 90 mM NaCl, 12 mM KCl, 25 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 10 mM sodium lactate (Kanto Chemical Co., Inc., Tokyo, Japan), and 10 mM Hepes was modified further by Suzuki [19] by the addition of 8 mM CaCl2, 2 mM sodium pyruvate (Sigma), 2 mM caffeine, and 5 mg/ml BSA (fraction V; Sigma). A portion (10 µl) of the preincubated spermatozoa was introduced into 90 µl of fertilization medium containing about 20 COCs surrounded by expanded cumulus cells. The final sperm concentration was adjusted to 1 x 105/ml. Coincubation was carried out at 39°C under 5% O2.
After coincubation of the gametes for 3 h, the oocytes were freed from the cumulus cells and attached spermatozoa and were transferred into IVC medium. The day of insemination was defined as Day 0. The basic IVC medium was NCSU-37 medium containing 4 mg/ml BSA and 50 µM ß-mercaptoethanol [20]. Two types of IVC medium were prepared: 1) basic medium with 5.55 mM D-glucose (Wako Pure Chemical Industries, Ltd., Osaka, Japan) (IVC-Glu), as originally reported [15], and 2) basic medium supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate (IVC-PyrLac). IVC was carried out at 38.5°C under 5% O2.
Embryo Evaluation
For examination of the IVF results, some inseminated oocytes were transferred to IVC-Glu and subsequently cultured for 7 h at 38.5°C under 5% O2. They were then fixed with acetic alcohol (1:3), stained with 1% aceto-orcein (Sigma), and examined for sperm penetration and pronuclear formation under a phase-contrast microscope. To examine their ability to develop to the blastocyst stage in vitro, all embryos and oocytes were cultured for 5 or 6 days, then fixed and stained. An embryo with a clear blastocele was defined as a blastocyst for the purposes of this study. The rate of blastocyst formation was evaluated, and the total number of cells in each blastocyst was evaluated as an indicator of embryo quality.
Estrous Synchronization
Estrous synchronization for supply of oviductal epithelial cells or for preparation of recipients was carried out as described previously [3]. In brief, an i.m. injection of 0.2 mg cloprostenol, prostaglandin F2
analogue (Planate; Sumitomo Seiyaku, Osaka, Japan) was given to pregnant gilts (7 mo old, 120–130 kg) on the 33rd to 53rd day of gestation, followed by a second injection of 0.2 mg cloprostenol 24 h later. One thousand IU of eCG (PMS 1000 Tani NZ) was administrated i.m. at the same time as the second cloprostenol injection. Ovulation was induced by i.m. injection of 500 IU hCG (Puberogen; Sankyo) 72 h after the eCG injection. Ovulation was expected to occur on the day of IVF at 41–42 h after the hCG injection.
Experiment 1
We evaluated the effects of oxygen concentration during IVM and energy supplementation during IVC on the ability of IVM-IVF oocytes to develop to the blastocyst stage. COCs were matured under 5% O2 or 20% O2 conditions. After IVF, IVC was carried out according to the following experimental design. IVP embryos were cultured in 1) IVC-Glu from Days 0 to 6 (medium was changed on Day 2); 2) IVC-Glu from Days 0 to 2, then IVC-PyrLac from Days 2 to 6; 3) IVC-PyrLac from Days 0 to 2, then IVC-Glu from Days 2 to 6; and 4) IVC-PyrLac from Days 0 to 6 (medium was changed on Day 2). The combined procedures of IVM and IVC gave a total of eight experimental groups for the study of developmental ability. After culture for 6 days, all embryos were fixed and evaluated.
Experiment 2
We investigated the effect of using conditioned medium (CM), produced from porcine oviductal epithelial cells, during the first 2 days of IVC on the ability of embryos develop to the blastocyst stage. Oviducts were collected from synchronized gilts on Day 0. They were washed several times with Dulbecco PBS (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) and cut into sections about 5 mm long. The surface of the epithelium was then exposed. The epithelial cells were planed with a surgical blade. The cell mass was suspended in PBS and washed three times by centrifugation at 600 x g for 2 min. The supernatant was discarded, and the cell mass was resuspended in IVC-PyrLac (3.4 x 106/ml). The cells were then cultured for 2 days at 38.5°C under 5% O2. After centrifugation, the supernatant was frozen at -20°C until use. After the supernatant had been thawed, an equal volume of fresh IVC-PyrLac was added, and the resultant solution was used as conditioned medium (IVC-PyrLac-CM).
IVM was carried out under 5% O2. IVF oocytes were cultured under the following regimens: 1) IVC-PyrLac from Days 0 to 2, then IVC-Glu from Days 2 to 6 and 2) IVC-PyrLac-CM from Days 0 to 2, then IVC-Glu from Days 2 to 6. All embryos were fixed on Day 6, and their development was evaluated after they had been fixed and stained.
Experiment 3
We evaluated the ability of IVM-IVF oocytes to develop to the blastocyst stage. IVM was carried out under 5% O2, and then IVF oocytes were cultured in IVC-PyrLac-CM from Days 0 to 2, followed by IVC-Glu until Days 5 or 6. Blastocysts categorized under a stereomicroscope as expanding or expanded were selected and fixed. All other embryos or oocytes were fixed. After they were stained, blastocysts that were neither expanding nor expanded could be correctly classified. The quality of all of the blastocysts was evaluated.
Experiment 4
We evaluated the ability of IVP blastocysts to develop to term after embryo transfer to recipients. IVM, IVF, and IVC were carried out as described for experiment 3. Blastocysts expanding on Day 5 and blastocysts that had expanded on Day 6 were transferred to IVC-Glu supplemented with 20 mM Hepes, the osmolarity of which had been adjusted to 285 osm/kg, as a transfer medium. Blastocysts that were expanding on Day 5 were surgically introduced into the uterus of a synchronized recipient on the fifth day after hCG injection, and blastocysts that had expanded on Day 6 were transferred to two recipients on the sixth day after the injection. A total of 50 blastocysts per recipient were transferred. All recipients were followed to term.
Statistical Analysis
Arcsine-transformed percentages of replications in rates of blastocysts [21], data for mean numbers of cells per blastocyst, and weights of newborn piglets were subjected to ANOVA by using the General Linear Models procedures of the Statistical Analysis System (SAS Institute Inc., Cary, NC) and were then analyzed by the Duncan multiple range test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There was no difference in the percentages of oocytes that matured and were penetrated by sperm between the conditions of 5% and 20% O2 during IVM, and almost all of the penetrated oocytes were activated, having both female and male pronuclei (Table 1). The percentages of monospermy also did not differ between the two oxygen concentration groups during IVM. These monospermic oocytes were judged as normally fertilized oocytes because they had single male and female pronuclei with a first and second polar body.
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Experiment 1
We evaluated the combined effects of oxygen concentration during IVM and energy supplementation during IVC on the ability of IVM-IVF oocytes to develop to the blastocyst stage (Table 2). The results of statistical analysis are shown in Tables 3, 4, and 5. Blastocyst formation rates did not differ between the two oxygen concentration groups (5% and 20% O2) during IVM. However, the total numbers of blastocyst cells differed between oxygen concentration groups (Table 3). Blastocysts developed from oocytes matured at the lower oxygen concentration had more cells than those matured at the higher oxygen concentration (Table 4). When IVF oocytes were cultured in four IVC systems with differing regimens of energy supplementation, significant differences were detected on statistical analysis (Table 3). The system in which embryos were cultured in IVC-PyrLac for the first 2 days followed by IVC-Glu for 4 days gave a higher rate of blastocyst formation than did the three other systems, and this system also had a significantly greater promoting effect on the total numbers of cells in the blastocysts (Table 5). Although we did not detect an interaction between the oxygen concentration and the IVC system (Table 3), we considered the combination of 5% O2 for IVM and IVC in IVC-PyrLac from Days 0 to 2 followed by IVC-Glu from Days 2 to 6 have excellent results. We therefore chose it as the basic IVP system for the subsequent experiments on the quality of blastocyst production.
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Experiment 2
When porcine oviductal cells were cultured, the oviductal epithelial cell mass demonstrated motility through the development of microvilli and began to form cystic structures. The motility and cystic cavity formation in the epithelial cells maintained well (Fig. 1) for about 3 wk, after which time the cells were cultured. CM was prepared on the second day in culture because, in vivo, porcine embryos are present in the oviducts until the four-cell stage (about 2 days after fertilization) [22]. When embryos were cultured in IVC medium supplemented with CM, although the rates of blastocyst formation were not significantly different, the total number of cells in the blastocysts on Day 6 was significantly higher than that in the blastocysts cultured in basic IVC medium (Table 6).
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Experiment 3
Blastocysts appeared after Day 5 of IVC. Expanding blastocysts on Day 5 and both expanded and expanding blastocysts on Day 6 could be selected under a stereomicroscope (Fig. 2). Other blastocysts with poor quality were defined after fixation and staining. All embryos were enclosed in zona pellucida and no hatching or hatched blastocyst appeared. After the embryos were fixed and stained, they were classified according to their developmental stages (Fig. 3), and we examined the rates of development of embryos to the blastocyst stage (Table 7) and the mean cell numbers of classified blastocysts (Table 8).
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Experiment 4
All recipients maintained their pregnancies, and they farrowed a total of 19 piglets (Table 9). The weights of newborn piglets were statistically different between Day 5 and 6 transfers. However, all of the piglets are showing normal growth.
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DISCUSSION |
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Our present results clearly demonstrate the ability of blastocysts obtained from prepubertal ovaries by using the modified IVP system to develop to term with excellent rates compared with those in the report of Marchal et al. [13], in which the transfer of IVP blastocysts to four recipients (14–26 blastocysts per recipient) resulted in one pregnancy and the birth of only two piglets. The main reasons for our success seem to be 1) the low oxygen concentration during IVM and IVF, 2) the use of IVC medium supplemented with pyruvate and lactate for the first 2 days of IVC, and 3) the conducting of IVC for the first 2 days in medium conditioned by oviductal epithelial cells.
To our knowledge, the precise effects of oxygen concentration during IVM and/or IVF of oocytes on their development to the blastocyst stage have not been reported previously in the pig. A low oxygen tension during IVM of bovine oocytes has been reported to be beneficial for supporting both maturation and blastocyst formation rates [26]. However, in the pigs in the present study, no significant difference in blastocyst formation rates was detected between the 5% O2 and 20% O2 IVM groups (Table 1). Nevertheless, after culture for 6 days, although the incidence of blastocyst formation was the same, the quality of blastocysts generated under 5% O2 IVM was better than that under 20% O2 IVM (Table 4). This suggests that a low O2 concentration during IVM improves embryo quality during IVC after IVF. Oxidative stress inducing apoptosis during pig IVM, which results in a low maturation rate, has been shown to be prevented by cumulus cells [27]. Although these researchers did not examine the relationship between oxidative stress during maturation culture and blastocyst quality after IVF, it was suggested that oocytes were exposed to oxidative stress during maturation culture. Therefore, the presence of a low O2 concentration can be considered to promote cytoplasmic maturation, leading to early embryonic development through a reduction of oxidative stress.
Our previous report [12] suggested that the low survival rate and poor quality of IVP blastocysts were caused by the use of an unsuitable IVC system, and also that the first 2 days of IVC have a critical effect on in vitro development. The first 2 days after IVF seem to be the prerequisite stage for embryonic genome activation [28], at which stage a four-cell block is observed in pigs. Our previous study [20] confirmed that porcine embryos produced in vitro differed in their timings of cleavage of blastomeres, resulting in a wide variety of developmental embryonic stages (two- to eight-cell stage at Day 2), but that most of them were at the late four-cell stage. Thus, it is probable that adequate chemical or oviductal supplements during the first 2 days may enhance embryonic potential for genome activation or for overcoming cell block. Therefore, we divided the IVC duration into two parts, the first 2 days (Days 0–2) and the following 4 days (Days 2–6), and examined the effects of 1) energy supplementation and 2) medium conditioned by porcine oviductal epithelial cells on the developmental potential to the blastocyst stage.
The need for energy sources to support embryo development in vitro was confirmed about 40 yr ago, suggesting the importance of lactate for culture of early mouse embryos [29, 30]. Pyruvate had also proven to be an important energy source for IVC of mouse early embryos [31, 32]. It is generally accepted that supplementation of pyruvate and lactate in the IVC medium is essential for successful IVC of embryos to the blastocyst stage in many species. Success in the IVC of porcine embryos produced in vivo to the blastocyst stage was reported with a medium containing both pyruvate and lactate [23–25]. On the other hand, a significant effect of glucose on IVC of in vivo one- or two-cell embryos to the blastocyst stage was reported [33]. NCSU-37 and -23 solutions [15], which are widely used for generating porcine IVP embryos, contain glucose but do not contain any pyruvate or lactate. To improve the IVC medium, we first added pyruvate or lactate instead of glucose to the NCSU-37-based IVC medium. The results clearly showed that there was a significant difference in the number of blastocyst cells between different energy supplementation regimens, although the rates of blastocyst formation were not significantly different (Table 5). Pyruvate and lactate seem to be important energy supplements in pigs, especially for early embryonic development in vitro. On the other hand, glucose seems to be necessary for the later part of IVC, because there was a higher rate of blastocyst formation with the use of IVC-Glu from Days 2 to 6 than with the use of IVC-PyrLac (Table 5). Our observation is confirmed by that in a previous report [34], which suggested that glucose is not metabolized readily by early porcine embryos before the eight-cell stage, but that it is used in higher amounts in embryos between the compacted morula and blastocyst stages. The detrimental effects of glucose during the early IVC period should be noted. It has been suggested that glucose is a source of two-cell block in the hamster [35]. In addition, the toxicity of methylglyoxal, a metabolic by-product of glycolysis, has been reported [36]; this by-product causes inactivation of the intracellular glutathione peroxidase that is responsible for scavenging oxygen free radicals [37]. Therefore, glucose supplementation may cause harmful effects, such as oxidative stress on genome activation or a difficulty in overcoming the four-cell block at about Day 2 of IVC in pigs.
Conditions closely approximating those in the oviduct are the best for porcine early embryos because the oviduct is the where fertilization occurs and where the embryo stays until the four-cell stage [22]. Therefore, the use of medium conditioned by oviductal epithelial cells would be expected to improve the IVP system. Cultivation in CM before IVF enhances in vitro embryonic development in pigs [38, 39], suggesting that CM contains a factor or factors secreted from oviductal epithelial cells. However, no effect of supplementation of CM after IVF on in vitro development to the blastocyst stage has been found [39]. In our study, CM also did not significantly accelerate blastocyst formation, but it did increase the number of cells per blastocyst after IVC for 6 days (Table 6). Although the mechanism of the improvement in blastocyst quality by CM is not clear at present, it is of interest that many kinds of secretory proteins have been reported to promote embryonic development: the most probable candidates are insulin-like growth factors 1 and 2 [40], activins [41, 42], glycoproteins [39, 43], and glycosaminoglycans [44, 45]. On the other hand, the concentrations of ions, such as those of calcium, magnesium, and potassium, vary with the stage of the estrous cycle [46]. Epithelial cells also secrete considerable amounts of amino acids [47] for the completion of early embryonic development [48, 49]. Besides the direct secretion of large molecules from the epithelial cells, it is possible that epithelial cells may modulate small-molecular substances during the preparation of CM. This may affect the developmental potential of the embryos after genomic activation at about Day 2.
Blastocyst formation was first observed on Day 5 during IVC. Some of the blastocysts were in an expanding state on Day 5, and their cell numbers and blastocele volume began to increase, resulting in the presence of expanded blastocysts on Day 6 (Table 7). The developmental stages of other blastocysts could be defined only after fixation and staining, because they could not be distinguished from fragmented embryos or oocytes before fixation and examination under a stereomicroscope. The total percentage of blastocysts was about 20% in each experiment, showing no difference between Days 5 and 6. This may suggest that the fate of IVP embryos is decided in the earlier days of IVC. We transferred both expanding blastocysts on Day 5 and expanded blastocysts on Day 6 to the recipients to check their ability to develop to term, and piglets resulted from all of these blastocysts. Vital embryo selection is possible on Days 5 and 6 by simple procedures (observation under a microscope). This procedure should be useful for predicting the viability of cloned embryos after nuclear transfer or for performing molecular analysis in transgenic embryos by saving recipients for embryo transfer. The average numbers of cells in expanding IVP blastocysts at Day 5 and expanded blastocysts on Day 6 were 49.7 and 80.2, respectively. The number of cells in blastocysts in vivo has been reported to be between 30 and 60, and it increases to about 175 just before hatching [50]. After these numbers are compared, IVP embryos appear to have smaller numbers than blastocysts in vivo, but some of them are nearly equal to those of in vivo blastocysts (e.g., the maximum number of cells in Day 6 expanded blastocyst was 164). These observations suggest that the IVP system in our study approximates the in vivo system well. Marchal et al. [13], in the first IVP experiments in piglets, reported that the mean number of cells in the blastocysts from prepubertal ovaries was 70. Although their IVP system differed from ours, the cell numbers of blastocysts in those studies seemed to be quite similar. Such a number of cells might be needed by blastocysts at Day 6 for successful piglet production after transfer to recipients.
The piglets farrowed from all of our recipients were quite normal. The weights of newborn piglets seemed to be the same as those from normal farrowings. However, the weights differed between the Day 5 and 6 transfers (Table 9), and the effect of the recipient was not significant (P = 0.14) after statistical analysis (data not shown). This calculation supports the possibility of differences in developmental potential depending on the transfer date. However, more replications are needed to confirm this, as we used only three recipients.
In conclusion, we established a successful IVP system for porcine IVM-IVF-IVC blastocysts and obtained healthy piglets after transfer of the blastocysts to the recipients. This system has advantages for the generation of cloned and transgenic pigs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: Kazuhiro Kikuchi, Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. FAX: 81 298 38 7408; kiku{at}nias.affrc.go.jp 
Accepted: November 5, 2001.
Received: August 20, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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