Classification of Morphological Changes Based on the Number of Cleavage Divisions in Bovine Embryos

Transcription

1 Journal of Reproduction and Development, Vol. 55, No. 1, 2009, Full Paper Classification of Morphological Changes Based on the Number of Cleavage Divisions in Bovine Embryos Hitoshi USHIJIMA 1,3), Kiyoshi AKIYAMA 2) and Toshio TAJIMA 3) Chiba Prefectural Ichihara Dairy Experimental Station, Chiba , 2) Kanagawa Prefectural Livestock Industry Technology Center, Ebina and 3) Chiba Prefectural Livestock Experimental Station, Yachimata , Japan Abstract. Quantification based on cleavage division (CD) of bovine preimplantation embryos facilitates quantitative analyses of embryonic developmental processes because CD occurs roughly once each day for all blastomeres for up to at least 9 days after ovulation. Therefore, embryonic morphological changes during this period were classified according to CD number. In this study, embryos collected from superovulated donors 0 9 days after ovulation were first classified morphologically into 14 conventional developmental stages. The total cell numbers (TCN) of embryos were measured using the air-dry method. The respective CD numbers of the embryos were then determined using logarithmic transformation of the TCN. The CD numbers of embryos were increased 0 10th with 11 stages. The 0th CD corresponded to 1-cell stage embryos; the 1st CD corresponded to 2-cell stage embryos; the 2nd CD corresponded to 3 4-cell stage embryos; the 3rd CD corresponded to 5 8-cell stage embryos; the 4th CD corresponded to 9 16-cell stage embryos, the 5th CD corresponded to morulae (17 32-cell stage embryos); and the 6th CD corresponded to the compact morulae. Furthermore, the 7th CD included early blastocysts to blastocysts. The 8th CD included expanded, collapsed and hatching blastocysts. The 9th CD included hatched blastocysts. The 10th CD included expanding-hatched blastocysts. The relationship between the CD number and the morphological characteristics of the bovine embryos 0 9 days after ovulation was expressed using a linear equation, and this revealed a high degree of correlation (y=0.98x 0.96, r=0.99). These results suggest that morphological changes of bovine embryos can be classified accurately using an 11- stage classification system based on the number of cleavages. Key words: Bovine, Cleavage division, Developmental stage, Embryo, Morphology, Total cell number (J. Reprod. Dev. 55: 83 87, 2009) Accepted for publication: October 15, 2008 Published online in J-STAGE: November 20, 2008 Correspondence: H Ushijima ( h.ushjm@ma.pref.chiba.lg.jp) efore implantation, mammalian embryos undergo characteristic morphological changes during development [1, 2]. Standard embryonic developmental stages have been defined retrospectively by observing embryo morphology based on the number of days after ovulation [3]. Additionally, in bovine developmental studies, the morphological characteristics of embryos are used for noninvasive examination as visual signs of embryo development [4 6]. Such morphological observations are widely used for research and practical purposes to evaluate embryonic developmental potency [7 9]. However, these type of observations are somewhat subjective; no clear standards have been established among laboratories [4 9]. Therefore, it is desirable to set an objective scale for accurate description of the developmental progress of bovine embryos. Morphological changes in mammalian embryos occur in close association with total cell numbers (TCNs) and number of cleavage divisions (CDs) [2, 10, 11]. At the same time, however, the segmental speed changes irregularly during development, being affected by cellular polarization and differentiation [2, 12 14]. Therefore, the relationship between CD number and morphological progress gradually weakens; for this reason, the CD number is not a favorable scale for measuring embryonic development for objective analysis. Van Soom et al. [15] suggested a strong association between CD timing and morphological changes not only in the precompaction stages but also in post-compaction stages of in vivoderived bovine embryos by showing that embryonic compaction and cavitation occur preferentially at the sixth and seventh cleavages, respectively. Recently, we demonstrated that cleavages, as calculated from the TCN of embryos, occur regularly once a day up to at least 9 days in high-quality, bovine embryos collected from superovulated donors [16]. That observation makes it possible to develop a standardized method for evaluating embryonic morphologies if a connection exists between the timing of morphological changes and the CD number. Herein, we describe analyses of the relationship between the morphology and number of cleavages of bovine embryos collected 0 9 days after ovulation. Furthermore, we assessed the conventionally defined developmental stages of bovine preimplantation embryos in terms of CD number. Materials and Methods Collection of in vivo-derived embryos All experiments were conducted using 43 cows and were designed individually and conducted with the approval of the animal experiment ethical committee of our institution. The embryos were collected from superovulated 3 8-year-old Holstein and Japanese black cows [17]. Thirty-four superovulated donors were distributed into groups of 3 5 donors for days 0 9 after ovulation (3 5 donors/day). In addition, 9 donors were used exclusively for embryo collection at the collapsed and hatching stage 7 8 days

2 84 USHIJIMA et al. Fig. 1. Examples of the 14 conventional developmental stages of bovine preimplantation embryos collected from superovulated donors. 1-cell, 2) 2-cell, 3) 3 4-cell, 4) 5 8-cell, 5) 9 16-cell (arrows indicate blastomeres at the 16-cell stage), 6) morula (arrows indicate blastomeres at the 32-cell stage), 7) compact morula, 8) early blastocyst (the arrow indicates the blastocoel), 9) blastocyst, 10) expanded blastocyst (the arrow indicates the thin zona pellucida), 1 collapsed blastocyst, 12) hatching blastocyst, 13) hatched blastocyst and 14) expanding-hatched blastocyst. after ovulation, at which embryos are difficult to find [5]. In brief, on Days 9 14 of estrus, follicle-stimulating hormone (FSH, Antrin; Kawasaki Mitaka, Tokyo, Japan) was given twice daily at decreasing doses (5/5, 4/4 and 3/3 mg) over 3 days. Doses of 7.5 mg tromethamine dinoprost (PG, Pronalgon F; Pfizer, Tokyo, Japan) at an interval of 6 h were administered twice to induce luteolysis 2 days after the first FSH treatment. The cows were observed for behavioral estrus and inseminated once 2.5 days after the first PG treatment. Ovulation after the superovulation protocol occurred about 0.5 days after the end of estrus; fertilization most likely occurred a few hours after ovulation [1, 3]. Embryos were recovered 0 3 days after ovulation by flushing each of oviducts with 20 ml of PBS containing 10% heat-inactivated fetal calf serum (FCS; Gibco BRL, Grand Island, NY, USA) after slaughter. Embryos were recovered nonsurgically from the uterine horns of cows 4 9 days after ovulation with 500 ml of the medium described above. Selection of embryos Many types of embryos at different developmental stages and various morphological qualities were collected from the superovulated donors, and the breed status of the donors [2, 15] affected neither embryo production nor developmental speed of bovine embryos. When high-quality embryos, which are selected according to morphology, are used, the TCNs of embryos can be counted accurately [11, 15, 16]. Therefore, the collected embryos were classified into five quality groups (excellent, good, fair, poor and degenerated) according to the morphological evaluation method [4] using an inverted phase-contrast microscope (IMT 10; Nikon, Tokyo, Japan). Excellent and good quality embryos were selected for use in the experiment and were classified into the following 14 conventional developmental stages: 1-cell; 2-cell; 3 4-cell; 5 8- cell; 9 16-cell, which is sometime called the morula or early morula; morula (M; the so-called cell stage, early morula, compacting morula or pre-compact morula); compact morula (CM; the so-called late morula or tight morula); early blastocyst (EB); blastocyst (B); expanded blastocyst (ExB); collapsed blastocyst (ColB); hatching blastocyst (HingB); hatched blastocyst (HB); and expanded hatched blastocyst (ExHB) stages [4 9, 15] (Fig.. Cell count of embryos and CD calculation Embryos were fixed using Tarkowski s air-dry method [18] with slight modification; they were stained with 5% Giemsa stain to

3 BOVINE EMBRYO DEVELOPMENT 85 Table 1. Total cell numbers (TCN) of bovine embryos in each developmental stage Developmental Collection days No. of embryos Cell number of embryos stage after ovulation examined Range Mean ± SD ± ± 0.0 * ± 0.3 * ± 1.2 * ± 1.8 * M ± 5.5 * CM ± 11.8 * EB ± 13.1 * B ± 11.2 * ExB ± 14.7 * ColB ± 24.1 HingB ± 15.0 * HB ± 55.2 ** ExHB ± 64.7 * M, morula; CM, compact M; EB, early blastocyst; ExB, expanded B; Col B, collapsed B; HingB, hatching B; HB, hatched B; and ExHB, expanding-hatched B. *, ** : Comparison of TCN with the preceding developmental stage: * P<0.001, ** P<0.01. allow counting of cells. In brief, embryos were treated with 0.9% sodium citrate solution for 5 40 min (5 min for embryos from the one-cell to pre-compact morula stage; 10 min for CM to HingB stage embryos; 20 min for HB stage embryos; and 40 min for ExHB stage embryos) at room temperature (RT) and were fixed onto a spot plate (Corning Labware and Equipment, Corning, NY, USA) using an ice-cooled fixative consisting of methanol, acetic acid and distilled water (3:2: for 1 min. They were then mounted on a glass slide using a small amount of fixative, air-dried for 1 h at RT, and stained with Giemsa stain (Gibco-Invitrogen, Carlsbad, CA, USA) for 10 min. According to the cell-count protocol [16], TCNs of the embryos were counted under a microscope at 400 magnification. The number of CDs (CDN) calculated from the TCNs of the embryos was expressible as an equation of the first degree with a high correlation to age (y=1.03x+0.16, r=0.99) [16]. This result means that all bovine blastomeres divide roughly once a day for up to 9 d after ovulation. Therefore, the CDN was calculated for the in vivo-derived embryos as log 2 of the TCN [11, 15, 19]. Finally, the relationship between CDN and morphological changes was analyzed statistically. Statistics The Student s t-test was used to compare the mean cell numbers of embryos. A probability of less than 0.05 was considered significant. The correlation between CDN and morphological stages was analyzed using the chi-squared test and simple linear regression to obtain a regression equation. This regression equation was compared to that for the conventional developmental stages using covariance analysis. Results In all, 122 embryos with lower numbers of degenerated blastomeres were used for the experiments. The mean TCNs of the bovine embryos are shown in Table 1 for the 14 conventional developmental stages. The TCN increased with morphological progress: there was a significant difference in TCN for each stage, except for ColB, compared with the preceding developmental stage (P<0.0. Table 2 presents the relationship between developmental stage and the calculated CDN. In regard to the early developmental stages, the precleavage stage (CDN=0) corresponded exclusively to 1-cell stage embryos, the 1st CD (CDN= corresponded to 2-cell stage embryos, the 2nd CD (CDN 2) corresponded to 3 4-cell stage embryos and the 3rd CD (CDN 3) corresponded to 5 8-cell stage embryos (P<0.00. Embryos that were classified morphologically as morulae were either at the 4th (9 16-cell stages), 5th (morula stages with more than 16 cells) or 6th CD (compact morula stage with more than 32 cells; P<0.00. Blastocysts, from the early to expanding-hatched stages, were divided into four CD stages: the 7th CD represented EB and B; the 8th CD represented ExB, ColB and HingB; the 9th CD represented HB; and the 10th CD represented ExHB with statistical significance (P<0.00. Consequently, the 14 developmental stages of the 0 9 d embryos were rearranged into the 11-class CDN system; the linear equation for these staging systems showed a high correlation (y= 0.98x 0.96, Pearson s correlation coefficient, r=0.99; Fig. 2). This result signifies that the morphological changes of developing bovine embryos were closely associated with the number of cleavage divisions. The regression line shows a higher correlation than that of the 14 developmental classifications vs. CDN (y= 0.74x+0.01, r=0.98; P<0.00. Discussion When bovine embryos were used selectively for bovine developmental studies after exclusion of low-quality embryos, the change in developmental speed was minimal and was different from in

4 86 USHIJIMA et al. Table 2. Relationship between the 14 conventional developmental stages and the number of cleavage divisions (CDN) in bovine preimplantation embryos collected from superovulated donors CDN Range of No. of embryos by the conventional classification Total P- cell No M CM EB B ExB ColB HingB HB ExHB embryos value P-value M, morula; CM, compact M; EB, early blastocyst; ExB, expanded B; ColB, collapsed B; HingB, hatching B; HB, hatched B; and ExHB, expandinghatched B. vitro-cultured embryos, in which developmental block and arrest are often observed [15, 16]. The results of our recent study also showed that cleavages occur at a constant interval in high-quality embryos until up to 9 days postovulation with a high correlation between the CD number and the number of embryonic days [16]. In the pre-morula stages of bovine embryos, morphological progress is closely related to the number of cleavages [2, 13, 19]. The results of the present study further demonstrate that morphological changes occur with a specific timeline that is definable by cleavage number from the 1-cell stage through the expanding hatched-blastocyst stage. We confirmed that the TCN increases with morphologic progress, as reported previously for the 14 conventional developmental stages (Table [2, 15]. In addition, the 11 stage classification system used for the present study shows higher correlation between the morphological transition and cleavage numbers (Table 2, Fig. 2). This result is consistent with the hypotheses advanced by Yang [10] and Van Soom et al. [15]. Herein, we propose this 11-stage system as a modified classification for describing morphological changes in bovine embryos: CDN0 represents the 1-cell stage, CDN1 represents the 2-cell stage, CDN2 represents the 3 4-cell stages, CDN3 represents the 5 8-cell stages, CDN4 represents the 9 16-cell stages, CDN5 represents the morula stages, CDN6 represents the compact morula stages, CDN7 represents the blastocyst stages, CDN8 represents the expanded blastocyst stages, CDN9 represents the hatched blastocyst stage and CDN10 represents the expanding-hatched blastocyst stage. Based on this CDN for bovine embryos, the numerical readout not only enables quantitative analysis of standard embryonic development, it also clearly represents the relationship between the morphological changes and cleavage numbers. Conventionally, researchers have divided morphological changes from the 9 16-cell to CM stages into two to five stages [4 9]. The results of the present study showed that three critical events take place during these stages. At the 4th CD stage, which comprises of 9 16-cell embryos, the embryonic genome becomes Fig. 2. Regression line of the CD number and 11-stage classification of bovine preimplantation embryos collected from superovulated donors: M, morula; CM, compact M; B, blastocyst; ExB, expanded B; HB, hatched B; and ExHB, expanding-hatched B. activated [13, 19]. During the 5th CD stage, containing morulae with more than 16-cells, superovulated embryos are transferred through the oviduct to the uterus [2, 10]. At the 6th CD stage, specialized junctions form between blastomeres [14]. Therefore, we believe that it is more sensible to divide this period into the following three stages based on these biological events: the 9 16-cell stage, M stage and CM stage. Embryos in the post-morula stage or later are most commonly classified according to when they are collected [7, 8]; embryos collected 6 days after ovulation are classified as CM or EB according to the presence or absence of the blastocoel. Similarly, embryos on Day 7 are divided into B or ExB according to the zona pellucida

5 BOVINE EMBRYO DEVELOPMENT 87 thickness, whereas those on Day 8 are classified into ColB, HingB or HedB depending on the progress of their hatching from the zona pellucida [4 9]. These classifications and naming, however, have not been standardized among different laboratories. The CDN classification, in contrast, divided blastocysts into 4 stages 6 9 after ovulation; the 7th CD is the blastocyst stage, during which cavitation starts. At the 8th CD, the blastocoel expands, thereby thinning the zona pellucida (ExB stage); at the 9th CD, the blastocyst hatches out, extruding itself from the zona pellucida. The 10th CD corresponds to the ExHB stage, during which the blastocyst is ellipsoidal. These developmental stages based on CD correspond well to characteristic morphological changes in post-morula stages. As described above, bovine embryos at the 9th CD are HB. However, most in vitro-cultured (IVC) bovine embryos become HB at the 8th CD [16]. Such a difference in the developmental progress of IVC embryos, in respect to the number of cleavages, has been reported previously [15]. It remains unclear why such irregularity occurs in IVC embryos and whether the hatched IVC blastocyst is normal morphologically. Further studies are necessary to examine IVC conditions to also maintain the close relationship between CDN and morphology in IVC embryos. This CDN classification system for bovine embryos will provide highquality criteria for IVC improvement. In conclusion, we demonstrated that bovine embryos undergo morphological changes that are closely correlated to the number of cleavage divisions up to at least 10 cleavages. This modified 11- stage classification system presents great potential as a standard for describing the preimplantation developmental stages of bovine embryos for research and practical purposes. References 1. Seidel GE Jr. Superovulation and embryo transfer in cattle. Science 1981; 211: Betteridge KJ, Flechon JE. The anatomy and physiology of pre-attachment bovine embryos. Theriogenology 1988; 29: Hamilton WJ, Laing JA. Development of the egg of the cow up to the stage of blastocyst formation. J Anatomy 1946; 80: Lindner GM, Wright RW Jr. Bovine embryo morphology and evaluation. Theriogenology 1983; 20: Donaldson LE. Day of embryo collection, quality and pregnancy rates in cattle. Vet Rec 1986; 118: Hasler JF, McCauley AD, Lathrop WF, Foote RH. Effect of donor-embryo-recipient interactions on pregnancy rate in a large-scale bovine embryo transfer program. Theriogenology 1987; 27: Seidel GE Jr, Seidel SM. Evaluation of embryos. In: Seidel GEJ, Seidel SM (eds.), Training Manual for Embryo Transfer in Cattle. Rome: FAO Technical Papers 77; 1991: Kanagawa H. Evaluation of embryos. In: Kanagawa H, Shimohira I, Saito N (eds.), Manual of Bovine Embryo Transfer. Tokyo: Jpn Livestock Tech Association; 1995: Robertson I, Nelson RE. Certification and identification of the embryo. In: Stringfellow DA, Seidel SM (eds.), Manual of the International Embryo Transfer Society, 3 rd ed. IL: IETS; 1998: Yang X. Embryo cloning by nuclear transfer in cattle and rabbits. Int Emb Trans Soc Newsletter 1991; 9: Papaioannou VE, Ebert KM. The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro. Development 1988; 102: Johnson MH, Maro B. Time and space in the mouse early embryo: a cell biological approach to cell diversification. In: Rossant J, Pedersen RA (eds.), Experimental Approaches to Mammalian Embryonic Development. Cambridge: Cambridge University Press; 1986: Barnes FL, Eyestone WH. Early cleavage and the maternal zygotic transition in bovine embryos. Theriogenology 1990; 33: Prather RS, First NL. A review of early mouse embryogenesis and its applications to domestic species. J Anim Sci 1988; 66: Van Soom A, Boerjan ML, Bols PEJ, Vanroose G, Lein A, Coryn M, de Kruif A. Timing of compaction and inner cell allocation in bovine embryos produced in vivo after superovulation. Biol Reprod 1997; 57: Ushijima H, Akiyama K, Tajima T. Transition of cell number in bovine preimplantation embryos from in vivo-collected and in vitro-produced embryos. J Reprod Dev 2008; 54: Eto T, Ishida K, Hayakawa S, Ushijima H. Superovulation of Japanese Black cattle with follicle stimulating hormone and human menopausal gonadotropin. Jpn J Anim Tech 1987; 9: (In Japanese). 18. Tarkowski AK. An air-dry-method for chromosome preparation from mouse eggs. Cytogenetics 1966; 5: Eyestone WH, First NL. Characterization of developmental arrest in early bovine embryos cultured in vitro. Theriogenology 1991; 35: