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Biology of Reproduction 64, 1375-1385 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Ultrastructural Morphometry of Bovine Blastocysts Produced In Vivo or In Vitro1

Adrienne E. Crosiera, Peter W. Farinb, Michael J. Dykstrab, Joseph E. Alexandera, and Charlotte E. Farin2,,c,a

a Departments of Animal Science, b Farm Animal Health and Resource Managment, and c Microbiology, Pathology, and Parasitology, North Carolina State University, Raleigh, North Carolina 27695-7621

ABSTRACT

The objective of this study was to compare the ultrastructure of bovine blastocysts produced in vivo or in vitro by using morphometric analysis. Blastocysts produced in vivo (multiple ovulations, MO) were obtained from superovulated Holstein cows. For blastocysts produced in vitro, cumulus-oocyte complexes aspirated from ovaries of Holstein cows were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into one of three culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi, followed by TCM-199 + 10% ECS from 72 to 168 hpi; and 3) mSOF (modified synthetic oviductal fluid): mSOF + 0.6% BSA. At 168 hpi, six or seven grade 1 blastocysts from each of the four treatments (MO, IVPS, IVPSR, and mSOF) were fixed and prepared for transmission electron microscopy. Random micrographs of each blastocyst were used to determine the volume density of cellular components. Overall, as blastocysts progressed in development, the volume densities of cytoplasm and intercellular space decreased (P < 0.05) and the volume densities of mature mitochondria, nuclei, blastocoele, and apoptotic bodies increased (P < 0.05). Across treatments, the proportional volumes of nuclei and inclusion bodies were increased in inner cell mass cells compared with trophectoderm cells for mid- and expanded blastocysts. For blastocysts produced in vitro, the volume density of mitochondria was decreased (P < 0.05) as compared with that of blastocycts produced in vivo. The proportional volume of vacuoles was increased (P < 0.05) in blastocysts from the mSOF treatment as compared with blastocysts produced in vivo. For mid- and expanded blastocysts from all three in vitro treatments, the volume density of lipid increased (P < 0.05) and the volume density of nuclei decreased (P < 0.05) compared with those of blastocysts produced in vivo. In conclusion, blastocysts produced in vitro possessed deviations in volume densities of organelles associated with cellular metabolism as well as deviations associated with altered embryonic differentiation. However, the specific nature of these deviations varied with the type of culture conditions used for in vitro embryo production.

early development, IVF/ART

INTRODUCTION

In vitro culture systems may result in embryos of reduced morphological quality compared with those produced in vivo [1–4]. Pregnancy rates following transfer of bovine blastocysts produced in vitro are lower than pregnancy rates following transfer of blastocysts produced in vivo [5]. This may be related to the reduction in agreement among evaluators observed when determining quality grade for embryos produced in vitro [4, 6]. Based on light-level assessment, embryos produced in vitro appear to have altered morphology, delayed development [7, 8], increased vacuolization, and appear more opaque as compared with embryos produced in vivo [9]. Furthermore, bovine blastocysts produced in vitro have fewer total cells as well as fewer inner cell mass cells [1], altered allocation of cells to the inner cell mass [3], and reduced cell-to-cell coupling as compared with blastocysts produced in vivo [10].

In vitro conditions for the development of bovine embryos vary from culture in the presence of serum to culture under serum-free conditions. Bovine blastocysts produced in serum-free medium had morphology comparable with that of blastocysts produced in vivo [11]. However, in vitro production of embryos in serum-free culture resulted in fewer blastocysts compared with culture in serum-supplemented medium [12]. The addition of serum to in vitro culture medium improved embryonic development in cattle [13–15] but prevented the definition of factors required for embryonic development [16]. Elimination of serum from culture medium for the first 72 h postinsemination (hpi) (serum restriction) resulted in embryos that were more advanced in development by 168 hpi but were of lower morphological quality compared with embryos produced entirely in the presence of serum [17].

Based on a qualitative study, the addition of serum to culture medium for ovine blastocysts resulted in an apparent increase in the number of lipid droplets and a decrease in mitochondrial number [18]. Culture in the presence of serum also may cause alterations in mitochondrial structure [19], which could compromise the ability of blastocysts to properly metabolize lipid [20]. Similarly, bovine blastocysts cultured in the presence of serum were reported to possess greater numbers of cytoplasmic vesicles and lipid droplets, as well as abnormal or immature mitochondria [2].

To date, ultrastructural characterization of bovine blastocysts has been based on qualitative observations. An objective morphometric analysis comparing the ultrastructural characteristics of preimplantation bovine blastocysts produced in vivo and in vitro has not been reported. Therefore, the overall objective of this study was to compare the ultrastructure of bovine blastocysts produced in vivo or in vitro using morphometric analysis. The following four specific objectives were pursued: 1) compare effects of stage of development (early-, mid-, or expanded blastocyst) on blastocyst ultrastructure, 2) assess ultrastructural differences between cell type (inner cell mass vs. trophectoderm) in mid- and expanded blastocysts, 3) examine the effect of embryo production systems (in vivo vs. in vitro) on blastocyst ultrastructure (early-, mid-, and expanded blastocysts combined), and 4) examine the effect of embryo production system on ultrastructure of mid- and expanded blastocysts only.

MATERIALS AND METHODS

Reagents and Hormones

Tissue culture medium (TCM-199 with Earle salts) was purchased from Gibco BRL (Grand Island, NY). Equine pituitary LH (11.5 NIH LH-S1 units/mg) and porcine pituitary FSH (50 mg/vial Armour FSH Standard) preparations were obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents and medium supplements were of tissue culture grade and were purchased from Sigma Chemical Co. Fatty acid-free BSA was purchased from Boehringer Mannheim (Indianapolis, IN). Low-melting-point agarose for embryo embedment was purchased from Bethesda Research Laboratories (Gaithersburg, MD). All other reagents for electron microscopy were purchased from Electron Microscopy Sciences (Fort Washington, PA). Prostaglandin F2{alpha} was obtained from Pharmacia & Upjohn Co. (Lutalyse; Kalamazoo, MI). FSH was obtained from Vetrepharm Canada (Folltropin; London, ON, Canada) or Ausa International (Super-OV; Tyler, TX).

In Vivo Embryo Production

For production of in vivo blastocysts (multiple ovulations, MO), Holstein cows were superovulated by i.m. administration of either 75 or 112.5 units of Super-OV or 300 mg Folltropin given in a series of decreasing doses over a 3- or 4-day period. Luteolysis was induced by i.m. administration of 25 mg prostaglandin F2{alpha} on the morning and evening of the third or fourth day of FSH treatment. Donor cows were artificially inseminated 12 and 24 h after first standing estrus with semen from a proven Holstein sire. Early-, mid-, and expanded-stage blastocysts were recovered by nonsurgical uterine flushing on Day 7 or Day 8 after first standing estrus (Day 0 = standing estrus). Over a period of 7 mo, a total of 33 grade 1 [21] blastocyst-stage embryos were recovered from six donor cows. Embryos were fixed in McDowell and Trump fixative (4% formaldehyde:1% glutaraldehyde; [22]) and held at 4°C. Seven individual blastocysts were then randomly selected from this pool and processed for transmission electron microscopy.

In Vitro Embryo Production

Cumulus oocyte complexes (COC) were aspirated, matured, and fertilized in vitro [23]. Ovaries from Holstein cows were collected at a local abattoir and held in saline with 0.75 µg/ml penicillin for 4–6 h. The COC were aspirated from 2- to 7-mm follicles and washed five times in modified Tyrode medium (TL-Hepes [24]). The COC were cultured in groups of 20–30 for approximately 22 h in TCM-199 with 10% heat-inactivated estrous cow serum (ECS), 10 µg/ml LH, 5 µg/ml FSH, 1 µg/ml estradiol, 200 µM pyruvate, and 50 µg/ml gentamicin. Cultures were maintained in an atmosphere of 5% CO2 in air with 100% humidity. At the end of the 22-h maturation period, COC were washed once and placed in fertilization medium consisting of heparin-supplemented Tyrode Albumin Lactate Pyruvate medium with 6 mg/ml fatty acid-free BSA [24]. Motile spermatozoa were obtained from the same Holstein sire used to produce embryos in vivo. Spermatozoa were selected by swim-up procedure [24] and used at a final concentration of 1 x 106 spermatozoa/ml. Gametes were coincubated for 18–20 h, after which time presumptive zygotes were washed six times in TL-Hepes. Zygotes, with their cumulus investments, were randomly distributed to one of three culture treatments for the 168-h embryo culture period. The treatments were 1) IVPS (in vitro produced with serum): TCM-199 with 10% ECS and 50 µg/ml gentamicin; 2) IVPSR (in vitro produced with serum restriction): TCM-199 with 1% (10 mg/ml) BSA and 50 µg/ml gentamicin until 96 hpi, followed by TCM-199 with 10% ECS and 50 µg/ml gentamicin from 96 hpi through 168 hpi; and 3) mSOF (modified synthetic oviductal fluid): mSOF (modified from Tervit et al. [25] and Lee and Fukui [26]) with 0.6% (6 mg/ml) BSA, 1% (v/v) minimal essential medium nonessential amino acids, 0.5 mM sodium citrate, 1.5 mM glucose, 0.33 mM pyruvate sodium salt, and 50 µg/ml gentamicin. All embryos were cultured in wells containing 1 ml of treatment medium in an atmosphere of 5% CO2 in air with 100% humidity. Culture media were changed at 48-h intervals throughout the 168-h culture period. Grade 1 blastocysts (n = 7, 6, and 7 for IVPS, IVPSR, and mSOF treatment groups, respectively) were harvested at 168 hpi, fixed in McDowell and Trump fixative, and held at 4°C until processing for transmission electron microscopy.

Electron Microscopy

Embedment procedures were modified from Dykstra [27]. Blastocysts were removed from fixative and washed three times for a total of 15 min in 0.1 M PBS. A solution of low-melting-point agarose (4% w/v) was prepared in 0.2 M PBS, and embryos were individually transferred into the liquid agarose at 37°C. The agarose was cooled to 25°C and allowed to harden. Agarose-embedded embryos were then removed with a razor blade as individual 3-mm3 blocks. Agarose blocks were postfixed in 2% osmium tetroxide in 0.2 M PBS for 45 min, washed three times in deionized water for a total of 15 min, dehydrated in an ethanol series, and individually embedded in Spurr resin [27]. Resin blocks were solidified at 80°C for at least 8 h. Ultrathin sections (80 nm) from each blastocyst were collected on copper grids and poststained with methanolic uranyl acetate and lead citrate [27]. Sections were visualized with a transmission electron microscope, and 7–14 random micrographs were obtained to represent each embryo. Micrographs were printed at a final magnification of 6720x. An average (± SEM) area of 46 346 ± 1288 µm2 was analyzed for each blastocyst or approximately 0.8% of the total volume.

Morphometry

General procedures The volume densities of cellular components were determined using the point-count method [28, 29]. A transparent grid consisting of 576 fine and 64 coarse test points was laid over each micrograph. The total number of points falling on cytoplasm was recorded for each blastocyst, and the volume density of cytoplasm was determined. This value was equivalent to the number of points falling on cytoplasm divided by the total number of points available on the test grid and was expressed as a percentage. The proportional volume, or volume density, occupied by each intracellular component was then calculated for each blastocyst as the number of points falling on a structure divided by the total number of points falling on the cytoplasm and was also expressed as a percentage. Within an individual embryo, the proportional volume of a given structure was computed based on the combined total counts from all micrographs for that embryo. Intracellular components assessed in this manner included mature, immature, vacuolated, and total mitochondria, lipid, vacuoles, nuclei, inclusion bodies, and apoptotic bodies. In addition, the cytoplasmic-to-nuclear ratio for each embryo was calculated as the total number of points falling on cytoplasm divided by the total number of points falling on nuclei.

The volume density occupied by each intercellular component was calculated as equivalent to the number of points falling on that structure divided by the total number of points available on the test grid. This value was expressed as a percentage, and the proportional volume of each component within an embryo was based on the combined total counts from all micrographs for that embryo. Intercellular components included intercellular spaces, blastocoele, debris within the blastocoele, extruded blastomeres, and all extruded material.

Mitochondria Three mitochondrial types were analyzed: 1) mature mitochondria, containing well-developed and evenly stacked cristae, 2) immature mitochondria, having poorly developed, peripheral cristae or a hooded appearance, and 3) vacuolated mitochondria, containing a membrane-bound vesicle. The volume densities for the three individual mitochondrial types were summed to represent the volume density of total mitochondria.

Statistical Analysis

Data for the volume densities for cellular components, cellular spaces, and debris were arcsine transformed and analyzed by General Linear Models procedures of SAS [30]. When a significant F-statistic was found, means were separated using the Duncan multiple-range test. Results are reported as least-squares means ± SEM. Means were considered statistically different at P < 0.05. Values between P = 0.055 and P = 0.10 are presented as tendencies (P < 0.10).

Statistical model 1 was used for analysis of the volume densities of intracellular components expressed as a proportion of cytoplasm and for all intercellular components. This model included the main effects of treatment and stage of blastocyst development (early-, mid-, and expanded blastocysts [21]).

Statistical model 2 was used for the analysis of intracellular components expressed as a proportion of cytoplasm for inner cell mass cells and trophectoderm cells from mid- and expanded blastocysts only. This model included effects of treatment, stage of blastocyst development, cell type (inner cell mass and trophectoderm), and the interaction of treatment by cell type.

Proportional data pertaining to the distribution of embryos between developmental stages among treatments and the proportional data pertaining to the occurrence of extruded material within blastocysts were analyzed using Categorical Data Analysis procedures (CATMOD) of SAS [30].

RESULTS

Types of mitochondria found within bovine blastocysts are depicted in Figure 1 [20]. Cellular components found within bovine blastocysts from the MO, IVPS, IVPSR, and mSOF treatments are illustrated in Figure 2, A–D, respectively. In Figure 3 is depicted the junction of inner cell mass (ICM) and trophectoderm (TE) cells for blastocysts produced in vivo (MO, Fig. 3A) or in vitro (IVPS, IVPSR, and mSOF, Fig. 3, B–D, respectively). Illustrations of both apoptotic bodies and inclusion bodies in bovine compact morulae have been previously published [20].



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  		FIG. 1. Examples of mitochondrial types found within bovine blastocysts (x24 000). A) Mature mitochondria (MM). Arrow indicates well-developed and evenly stacked cristae. B) Immature mitochondria containing few peripheral cristae (IM) or having a hooded appearance (HM). C) Vacuolated mitochondrion (VM) containing a membrane-bound vacuole (arrow) within the organelle



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  		FIG. 2. Representative sections of inner cell mass (ICM) from bovine mid- and expanded blastocysts. A) Sections through the ICM of a blastocyst produced in vivo (x4800). B–D) Blastocysts produced in vitro from IVPS (B), IVPSR (C), and mSOF (D) treatments (x4800). Examples of lipid droplets (L), vacuoles (V), mitochondria (M), nuclei (N), and intercellular spaces (IS) are indicated



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  		FIG. 3. Junction of ICM and trophectoderm (TE) in mid- and expanded-stage bovine blastocysts. A) Mid-blastocyst produced in vivo (MO, x3600). B–D) Mid-blastocysts produced in vitro from IVPS (B), IVPSR (C), and mSOF (D) treatments (x3600). Examples of ICM, TE, mitotic figure (MF), apoptotic body (AB), blastocoele (B), microvilli (*), zona pellucida (ZP), extruded blastomeres (EB), and extruded degenerate material (EM) are indicated

Effect of Developmental Stage on Volume Density of Cellular Components in Bovine Blastocysts

The distribution of embryos between the three stages of development did not differ among the four treatment groups (P = 0.96). The numbers of embryos for early-, mid-, and expanded-blastocyst stages by treatment were 2, 3, and 2 for MO; 2, 3, and 2 for IVPS; 2, 1, and 3 for IVPSR; and 2, 2, and 3 for mSOF, respectively. Because of low subclass numbers, the interaction of treatment by stage of development could not be included in the final statistical model. Therefore, only the main effects of treatment and stage of development are presented. The data in Table 1 represent the effect of stage of development on the volume densities of cellular components. Data for individual early blastocysts represent the proportional volume of cellular components within the one undifferentiated cell type. Data for mid- or expanded blastocysts represent combined values for the volume densities of cellular components for the ICM and TE cell types.


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  		TABLE 1. Volume density (%*) of cellular components in bovine blastocysts at different stages of development

The proportional volume of cytoplasm was reduced (P < 0.05) in mid- and expanded blastocysts as compared with early blastocysts (Table 1). No differences were found among stages of blastocyst development for the volume densities of total, immature, or vacuolated mitochondria. However, there was an increased (P < 0.05) proportional volume of mature mitochondria in mid- and expanded blastocysts as compared with early blastocysts. There was no difference in the proportional volumes of vacuoles or lipid by stage of blastocyst development (Table 1).

There was an increased (P < 0.05) proportional volume of nuclei in expanded blastocysts as compared with early blastocysts. As a result of the decreased proportional volume of cytoplasm and the increased volume density of nuclei, the cytoplasmic-to-nuclear ratio was decreased (P < 0.05) in expanded blastocysts as compared with early blastocyst stage embryos (Table 1). As expected, the proportional volume of blastocoele increased (P < 0.05) with developmental stage. There was a decreased (P < 0.05) proportional volume of intercellular space in mid- and expanded blastocysts as compared with early blastocysts. There was no effect of stage of development on the volume densities of either debris within the blastocoele or inclusion bodies. However, the proportional volume of apoptotic bodies was decreased at the early-blastocyst stage compared with mid- and expanded blastocysts (Table 1).

Effect of Cell Type on Volume Density of Cellular Components in ICM and TE Cells of Mid- and Expanded Blastocysts Only

No significant interactions of treatment by cell type were found for any of the components analyzed. Therefore, only the main effects of treatment and cell type, each adjusted for stage of development, are presented. There were no differences in the proportional volumes of total, mature, immature, or vacuolated mitochondria between ICM and TE cell types. In addition, there was no difference in the proportional volumes of lipid, vacuoles, or apoptotic bodies between the two cell types (Table 2). The volume density of inclusion bodies in cells of the ICM was increased (P < 0.05) as compared with cells of the TE. The volume density of nuclei was greater (P < 0.05) in cells of the ICM than in cells of the TE (Table 2).


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  		TABLE 2. Volume density (%*) of cellular components in ICM and TE cells of mid- and expanded-stage bovine blastocysts

Effect of Treatment on Volume Density of Cellular Components in Bovine Blastocysts Across All Stages of Development

Table 3 and Figure 4 contain data on the effect of embryo production system (MO, IVPS, IVPSR, and mSOF) on the proportional volumes of cellular components combined across all three stages of development. The volume densities of lipid in blastocysts from the IVPSR and mSOF treatments were greater (P < 0.05) than in blastocysts produced in vivo (Fig. 4A). The proportional volume of vacuoles was greater (P < 0.05) in blastocysts produced in mSOF than in blastocysts produced in vivo or in the IVPS treatment (Fig. 4B).


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  		TABLE 3. Volume density (%*) of cellular components in bovine blastocysts produced under different treatment conditions



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  		FIG. 4. A) Volume density of lipid in bovine blastocysts from MO, IVPS, IVPSR, and mSOF treatments (statistical model 1, a,bP < 0.05). B) Volume density of vacuoles in bovine blastocysts from MO, IVPS, IVPSR, and mSOF treatments (statistical model 1, a,bP < 0.05)

Blastocysts from the IVPSR treatment group had an increased (P < 0.05) proportional volume of cytoplasm as compared with blastocysts produced in vivo or in mSOF (Table 3). There was a decreased (P < 0.05) volume density of total mitochondria for blastocysts produced in vitro as compared with those produced in vivo (Table 3). Although there was no effect of treatment on the volume density of mature mitochondria, the proportional volumes of both vacuolated and immature mitochondria tended (P < 0.10) to be decreased in blastocysts from all three in vitro treatments compared with those produced in vivo.

Blastocysts from the IVPS and IVPSR treatment had a reduced volume density of nuclei as compared with blastocysts produced in vivo (Table 3). The proportional volume of apoptotic bodies tended (P < 0.10) to be increased for all blastocysts produced in vitro as compared with those produced in vivo. There was no effect of treatment on the proportional volumes of inclusion bodies, debris, blastocoele, or intercellular space. Blastocysts from the IVPSR treatment had an increased cytoplasmic-to-nuclear ratio (P < 0.05) as compared with that for blastocysts produced in vivo (Table 3).

Effect of Treatment on Volume Density of Cellular Components in Mid- and Expanded Bovine Blastocysts

As blastocysts develop, the blastocoele increases in size and cells become organized into the ICM and TE. Coincident with this reorganization, the demand for ATP and the consumption of metabolic substrates also increases [31]. As a result, the volume density of cellular components may be altered in response to the physiological changes that occur between the early- and mid- to expanded-blastocyst stages. Therefore, we evaluated the subset of mid- and expanded-stage blastocysts for effects of culture treatment on cellular organelles. Table 4 and Figure 5 contain data on the effect of embryo production system (MO, IVPS, IVPSR, and mSOF) on the volume densities of cellular components for mid- and expanded blastocysts combined across cell type (ICM and TE).


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  		TABLE 4. Volume density (%*) of cellular components in mid- and expanded-stage bovine blastocysts produced under different treatment conditions



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  		FIG. 5. A) Volume density of lipid in mid- and expanded-stage bovine blastocysts from MO, IVPS, IVPSR, and mSOF treatments (statistical model 1, a,bP < 0.05). B) Volume density of vacuoles in mid- and expanded-stage bovine blastocysts from MO, IVPS, IVPSR, and mSOF treatments (statistical model 1, a,bP < 0.05)

The volume density of lipid was greater (P < 0.05) for mid- and expanded blastocysts produced in vitro as compared with those produced in vivo (Fig. 5A). There was an increased (P < 0.05) proportional volume of vacuoles for mid- and expanded-stage blastocysts produced in mSOF as compared with those produced in vivo or in the IVPS or IVPSR treatments (Fig. 5B).

There was no effect of treatment on the proportional volume of cytoplasm for mid- and expanded blastocysts (Table 4). However, the volume density of total mitochondria was decreased (P < 0.05) for blastocysts from all three in vitro treatments as compared with that for those produced in vivo (Table 4). There was no effect of treatment on the proportional volume of mature mitochondria. Blastocysts from all three in vitro treatments tended (P < 0.10) to have decreased volume densities of both immature and vacuolated mitochondria as compared with that for blastocysts produced in vivo.

There was no effect of treatment on the volume density of apoptotic bodies (Table 4). However, blastocysts from the IVPS and mSOF treatments had decreased (P < 0.05) proportional volumes of inclusion bodies as compared with those produced in vivo. The proportion of cytoplasm occupied by nuclei was decreased (P < 0.05) in blastocysts produced in vitro as compared with that for those produced in vivo (Table 4). The cytoplasmic-to-nuclear ratio tended (P < 0.10) to be increased for blastocysts from all in vitro treatments as compared with that for those produced in vivo. Blastocysts from the mSOF treatment tended (P < 0.10) to have an increased volume density of intercellular space as compared with blastocysts produced in vivo or with the IVPS treatment (Table 4).

The volume density of extruded blastomeres was increased (P < 0.05) in mid- and expanded blastocysts from the IVPSR treatment as compared with those produced in vivo (Table 4). There was no effect of treatment on the volume density of all extruded material. However, 2 of the 5 (40%) blastocysts produced in vivo (MO) possessed extruded material, whereas 12 of the 14 (85.7%) blastocysts produced in vitro (IVPS, IVPSR, and mSOF combined) possessed extruded material (P = 0.06). There was no effect of treatment on the proportional volume of either blastocoele or debris within the blastocoele. The ICM-to-TE ratio, calculated based on relative ratio of cytoplasm present for each cell type, tended (P < 0.10) to be lower for blastocysts from the IVPS and IVPSR treatments than for those from in vivo (MO) controls (Table 4).

DISCUSSION

Ultrastructural Changes with Stage of Blastocyst Development

Based on morphometric analysis, cellular components changed in a manner consistent with that which might be predicted for bovine embryos progressing through the blastocyst stages. As these embryos advanced in development, the proportional volume of intercellular space decreased as the blastocoele became more organized and occupied an increased proportion of the embryo. The accumulation and composition of fluid in the blastocoele during early embryonic development is regulated by Na/K-ATPase activity in mouse embryos [32] and is essential for differentiation of the ICM and TE cell types [33]. As embryos progress in development from early to expanded stages, cytoplasm would be expected to occupy a decreasing proportion of the entire blastocyst as a result of increased fluid accumulation within the blastocoele and subsequent flattening of embryonic cells [34]. This prediction is consistent with observations in the present study. Furthermore, the increased proportional volume of nuclei in blastocysts as they advanced in development in the present study is consistent with the observation that cell number increases significantly between the early- and expanded-blastocyst stages of development [1]. Concurrent with these morphometric changes, the cytoplasmic-to-nuclear ratio decreased as blastocysts advanced from the early to expanded stage.

The cytoplasm of bovine embryos produced in vivo or in vitro contains numerous lipid droplets prior to the blastocyst stage [35]. As embryos develop, excess lipid may be sequestered within the cell and utilized by mitochondria for increased production of ATP [36, 37] required for blastocoele formation or the differentiation of cell lineages. In the present study, no differences were found in the proportional volume of lipid across developmental stages or between cells of the ICM and TE. This finding may be an indication that lipid is continually sequestered throughout blastocyst development.

It has been suggested that mitochondrial structure is altered throughout bovine preimplantation development [35] and follows a progression from electron-dense organelles in early cleavage stages to organelles with multiple central cristae at the compact morula stage [38]. In addition, the matrix of mitochondria found within murine morulae and blastocysts had an increased amount of transverse cristae compared with those found within early cleavage stage embryos [39]. The increased volume density of mature mitochondria observed for embryos as they progressed through the stages of blastocyst development is consistent with these observations.

Cell death and degeneration are found in mammalian preimplantation embryos produced in vivo [40, 41] and may be important for normal embryonic development [42]. Although there was no difference in the proportional volume of apoptotic bodies between cells of the ICM and TE, there was an increased proportional volume of apoptotic bodies as blastocysts advanced in development. This may be an indication that embryos possess the ability to remove cells with reduced developmental potential [41]. The increased volume density of inclusion bodies found in cells of the ICM compared with cells of the TE may also be an indication that embryos are able to remove cells with inappropriate potential [43] and regulate cell number during embryonic differentiation [44].

Effects of Treatment on Blastocyst Ultrastructure

Compared with bovine embryos produced in vivo, embryos produced in vitro possess ultrastructural differences [2, 20, 35] that may result from the specific culture system used for embryo production [45, 46]. An increased volume density of lipid was seen in mid- and expanded blastocysts from the three in vitro culture treatments as compared with those produced in vivo. This is consistent with our previous observations that compact morulae produced in vitro in either serum-supplemented or serum-free medium contained an increased proportional volume of lipid as compared to morulae produced in vivo [20]. An increased amount of lipid in embryos cultured in vitro was suggested to result from the uptake of lipid from serum included in the culture medium [18, 47]. It is more likely, however, that lipid accumulation is a result of insufficient metabolism by mitochondria within embryos produced using any of these culture systems. The observed reduction in the volume density of total mitochondria for blastocysts from the in vitro production systems is consistent with this hypothesis. Mitochondrial function may be predictive of developmental potential of bovine embryos [48]. Thus, the concurrent increase in lipid and decrease in mitochondria within these in vitro-produced embryos may have a negative impact on subsequent embryonic development.

For blastocysts analyzed in the present study, there was no difference between treatments in the proportional volume of the blastocoele. Thus, the establishment of cellular mechanisms required for fluid accumulation during embryonic development may not be compromised by in vitro culture. However, the increased volume density of intercellular space present in the mid- and expanded blastocysts from the mSOF treatment is consistent with reduced cell-to-cell coupling observed in bovine blastocysts produced in vitro as compared with those produced in vivo [10]. This may indicate a lesser degree of organization present in these blastocysts produced in the serum-free medium as compared with those produced in vivo.

Cytoplasmic vacuoles are present in bovine [34, 35] and ovine [49] embryos produced in vivo and become fewer in number as development progresses. However, the functional capacity of these vacuoles within embryonic cells remains unknown. Cytoplasmic vacuoles may function to store glycogen in bovine oocytes [50]. Alternatively, cytoplasmic vacuoles may result from phagocytosis of material from cell autolysis in bovine [34] and primate [42] blastocysts produced in vivo. For embryos in the present study, there was no difference in the volume density of vacuoles as embryos progressed from the early-blastocyst to the mid- and expanded-blastocyst stages. In addition, there was no difference in the proportional volume of vacuoles in cells of the ICM as compared with cells of the TE. However, blastocysts from the mSOF treatment had an increased volume density of cytoplasmic vacuoles as compared with blastocysts from all other treatments. This may be indicative of abnormal [41] or delayed [2] developmental differentiation of blastocysts from this serum-free culture medium.

The observed reduction in the proportional volumes of nuclei and the increased cytoplasmic-to-nuclear ratio for mid- and expanded blastocysts from all three in vitro treatments are consistent with reports that blastocysts produced in vitro undergo fewer cellular divisions than do those produced in vivo [51]. In addition, in vitro culture of bovine blastocysts altered differentiation and reduced the number of ICM cells as compared with production of embryos in vivo [3, 44]. In the present study, the decreased ratio of ICM-to-TE cell cytoplasm for blastocysts from the IVPS and IVPSR treatments as compared with blastocysts produced in vivo may be an indication that fewer ICM cells are present relative to the number of TE cells in these blastocysts. Taken together with the observation that bovine embryos produced in vitro reach the blastocyst stages of development at a lower cell number than their in vivo counterparts [51], these observations may have negative implications for pregnancy rates following transfer of embryos produced in vitro.

Trophoblast cells of bovine blastocysts produced in vivo have well-developed microvilli projecting into the perivitelline space, whereas embryos considered to be morphologically deviant may be almost completely devoid of microvilli [34]. Compared with bovine blastocysts produced in vivo, blastocysts produced in vitro had shorter and less numerous microvilli on the apical membrane of trophoblast cells and may, therefore, posses a reduced absorptive capacity [2]. In the present study, blastocysts produced in vitro often lacked a well-developed and well-organized TE layer with accompanying microvilli projecting into the perivitelline space (Fig. 3, A vs. C). Because differential staining relies on the presence of an intact TE layer [52], the accurate assessment of ICM and TE cell numbers may be compromised for embryos produced in vitro [1, 53].

There were no differences among treatment groups in the volume density of all extruded material. This included the volume densities of extruded blastomeres and extruded degenerate material within the perivitelline space. However, 40% of blastocysts produced in vivo (MO) possessed extruded material as compared with 86% of blastocysts produced in vitro (incidence for IVPS, IVPSR, and mSOF of 60%, 100%, and 100%, respectively). A dark vacuolar appearance has been associated with bovine blastocysts produced in the presence of serum and was suggested to result from the uptake of lipid from the culture medium [45]. However, the presence of extruded material in these blastocysts could also result in a dark vacuolar or patchy appearance (see Fig. 3B) and could possibly be mistaken for an ICM when embryos produced in vitro are examined with light microscopy only (see Fig. 3D).

In summary, as blastocysts progressed in development, the volume densities of cytoplasm and intercellular space decreased and the volume densities of mature mitochondria, nuclei, blastocoele, and apoptotic bodies increased. The proportional volume of vacuoles was increased in blastocysts from the mSOF treatment as compared with blastocysts produced in vivo. For mid- and expanded blastocysts from all three in vitro treatments, the volume density of lipid increased and the volume densities of mitochondria and nuclei decreased as compared with those of blastocysts produced in vivo. In conclusion, blastocysts produced in vitro possessed deviations in volume densities of cellular organelles associated with cellular metabolism and possessed multiple deviations associated with altered embryonic differentiation. However, the specific nature of these deviations varied with the type of culture conditions used for in vitro embryo production.

ACKNOWLEDGMENTS

The authors acknowledge Brendalyn Bradley-Kerr for assistance in specimen preparation, identification of cellular components, and preparation of publication photographs. The authors also thank Drs. John Gadsby and William Flowers for critical review of this manuscript.

FOOTNOTES

First decision: 22 August 2000.

1 Supported by USDA Grant 9602482 and the North Carolina Agricultural Research Service. Back

2 Correspondence: Charlotte E. Farin, Department of Animal Science, North Carolina State University, Box 7621, 231B Polk Hall, Raleigh, NC 27695-7621. FAX: 919 515 7780; char_farin{at}ncsu.edu Back

Accepted: December 12, 2000.

Received: July 6, 2000.

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