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Biology of Reproduction 62, 1459-1465 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Ultrastructural Morphometry of Bovine Compact Morulae Produced In Vivo or In Vitro1

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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this study was to compare the ultrastructure of bovine compact morulae produced in vivo or in vitro using morphometric analysis. Compact morulae produced in vivo were obtained from superovulated Holstein cows. Compact morulae produced in vitro were obtained from cumulus-oocyte complexes aspirated from ovaries of Holstein cows. The complexes were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into 1 of 3 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 144 hpi; 3) mSOF (modified synthetic oviductal fluid): SOF + 0.6% BSA. At 144 hpi, five grade 1 compact morulae from each of the four treatments were prepared for transmission electron microscopy. The volume density occupied by cellular components was determined by the point-count method using a sampling of seven to nine random micrographs from each compact morula. The volume density of lipid was greater (P < 0.05) in compact morulae from IVPS, IVPSR, and mSOF treatments compared with those produced in vivo. There was a reduced proportional volume of total mitochondria in compact morulae from the IVPS treatment compared with those produced in vivo (P < 0.05). For compact morulae from the IVPS culture treatment, the volume density of vacuoles was greater than that for compact morulae produced in vivo (P < 0.05). The cytoplasmic-to-nuclear ratio for compact morulae from the IVPS treatment was increased (P < 0.05) compared with the ratio for those produced in vivo. In conclusion, compact morulae produced in vitro differed ultrastructurally from those produced in vivo. Compact morulae produced in IVPS culture medium possessed the greatest deviations in cellular ultrastructure.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Compaction of morula-stage mammalian embryos is required for proper blastocyst formation [1] and leads to differentiation of the inner cell mass and trophectoderm [2]. Many structural events are associated with compaction, including blastomere polarization, blastomere flattening [1, 3, 4], and gap junction formation [2]. In bovine and human embryos, the events of compaction are dependent on embryonic age, blastomere number, and number of cell divisions [5, 6].

Bovine embryos produced in vitro can be characterized as having altered morphology compared with those produced in vivo. Compared with embryos produced in vivo, embryos produced in vitro have fewer total blastomeres [7], demonstrate incomplete compaction [8, 9] and cell-to-cell coupling [10], and have a greater variance in morphological quality and developmental rate [11]. Differences in morphology may contribute to the decrease in agreement among evaluators when determining stage of development for compact morulae produced in vitro [12, 13]. Furthermore, it is interesting to note that pregnancy rates after transfer of morula-stage bovine embryos produced in vitro [14] were lower than pregnancy rates after the transfer of morulae produced in vivo [15].

Efforts to improve methods for the production of embryos in vitro are dependent on a better understanding of the effects of culture environments on embryo development [16]. It has been suggested that addition of serum to culture medium increases the occurrence of lipid droplets [17, 18] and alters mitochondrial structure in ovine embryos [19]. Elimination of serum from culture medium for the first 72 h postinsemination (hpi) resulted in bovine embryos that were more advanced in development by 168 hpi but were of lower morphological quality than those produced entirely in the presence of serum [20]. Embryos produced in serum-free medium possessed fewer lipid droplets and vacuoles compared with those cultured in the presence of serum [18, 21].

To date, ultrastructural evaluations of preimplantation-stage embryos from sheep and cattle have been based on subjective observation [16–19, 21–24]. Morphometric analysis offers a more objective method of assessing differences in cellular ultrastructure [25–29] that may occur in embryos as a result of in vitro culture. Therefore, the objective of this study was to use morphometric analysis to quantify the ultrastructure of bovine compact morulae produced either in vivo or in vitro using three embryo culture media.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Hormones

Tissue culture medium (TCM-199 with Earle's Salts) was purchased from Gibco-BRL (Grand Island, NY). Equine pituitary LH (11.5 NIH LH-S1 U/ml; Bethesda, MD) and porcine pituitary FSH preparations were obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents and media supplements were of tissue culture grade and were purchased from Sigma. 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 (Lutalyse; Kalamazoo, MI). Folltropin was obtained from Vetrepharm Canada (London, ON, Canada).

In Vivo Embryo Production

For production of in vivo compact morulae (multiple ovulations, MO), Holstein donor cows were superovulated by i.m. administration of 400 mg Folltropin given in a series of decreasing doses over a 3- or 4-day period. Estrus 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. Estrus detection was performed twice daily beginning 24 h after the first prostaglandin F2{alpha} injection. Donor cows were artificially inseminated 12 and 24 h after first standing estrus with semen from a proven Holstein sire. Embryos used in this study were recovered by nonsurgical uterine flushing of donor cattle on either Day 6 or Day 8 of the cycle (Day 0: first standing estrus). Based on evaluation at x60, a total of five grade 1 [30] compact morulae were identified, fixed in McDowell's and Trump's fixative (4% formaldehyde:1% glutaraldehyde; [31]), and held at 4°C until processed for transmission electron microscopy.

In Vitro Embryo Production

Cumulus-oocyte complexes (COC) were aspirated, matured, and fertilized in vitro as described by Farin and Farin [32]. Briefly, ovaries from Holstein cattle were collected at a local abattoir and held in saline with 0.75 µg/ml penicillin for 4–6 h at ambient temperature. COC were aspirated from 2- to 7-mm follicles and washed five times in modified Tyrode's medium (TL-Hepes; [33]). COC were matured in groups of 20–30 for approximately 22 h in 1 ml 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 sodium salt, 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 TALP medium with 6 mg/ml fatty acid-free BSA [33]. Thawed frozen semen from the same Holstein sire was used for production of all in vivo and in vitro embryos. Motile spermatozoa were collected by swim-up procedure [33] 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 and randomly distributed to one of three culture treatments. These 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 10 mg/ml BSA and 50 µg/ml gentamicin until 72 hpi followed by TCM-199 with 10% ECS and 50 µg/ml gentamicin from 72 hpi to 144 hpi; 3) mSOF (modified synthetic oviductal fluid): synthetic oviductal fluid with 6 mg/ml BSA, 1% v:v MEM nonessential amino acids, 0.5 mM sodium citrate, 1.5 mM glucose, 0.33 mM pyruvate sodium salt, and 50 µg/ml gentamicin (modified from Tervit et al. [34] and Lee and Fukui [35]). All embryos were cultured in wells containing 1 ml treatment medium in an atmosphere of 5% CO2 in air with 100% humidity. Culture media were changed at 48-h intervals throughout the 144-h culture period. Grade 1 [30] compact morulae (n = 5 per treatment) were harvested at 144 hpi, fixed in McDowell's and Trump's fixative, and held at 4°C until processed for transmission electron microscopy.

Electron Microscopy

Embedment procedures were modified from Dykstra [36]. Compact morulae 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 as individual 3-mm3 blocks with a razor blade. 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, subjected to an alcohol dehydration series, and individually embedded into Spurr's resin [36]. Resin blocks were solidified at 80°C for at least 8 h. Ultrathin sections (80 nm) from each compact morula were collected onto copper grids and poststained with methanolic uranyl acetate and lead citrate [36]. Sections were visualized on a transmission electron microscope, and 7 to 9 random micrographs were taken to represent each embryo. Micrographs were printed at a final magnification of x6720. An average of 37 534 ± 773 µm2 (mean ± SEM) was analyzed for each compact morula.

Morphometry

General procedures The volume density of cellular components was determined utilizing the point-count method [29]. Briefly, a transparent grid consisting of 576 fine and 64 coarse test points was laid over each micrograph. The number of test points falling on an individual structure was recorded, as was the total number of test points available on the test grid [26, 29]. The volume occupied by each component was equivalent to the number of points falling on that structure divided by the total number of test points available on the test grid. This figure was then expressed as a percentage. Within an individual embryo, the volume density, or proportional volume, was computed based on the combined total counts from all micrographs for that embryo.

Mitochondria Three types of mitochondria were analyzed: 1) mature mitochondria, containing well-developed and evenly stacked cristae [37–39]; 2) immature mitochondria, having poorly developed, peripheral cristae or a hooded appearance [22, 37]; and 3) vacuolated mitochondria, containing a membrane-bound vesicle [38]. The volume density of total mitochondria was calculated based on the sum of the densities for the three individual mitochondrial types.

Other cellular components The volume densities of lipid droplets, vacuoles, inclusion bodies, apoptotic bodies, intercellular space, debris between cells, nuclei, and cytoplasm, as well as the cytoplasmic-to-nuclear ratio, were determined.

Statistical Analysis

The volume densities for all cellular and intercellular components were analyzed by General Linear Models procedures of the Statistical Analysis System [40]. When a significant F-statistic was found, treatment differences were identified using Duncan's multiple range test. Effects of treatment were considered statistically different at P < 0.05. All data are reported as least-squares means ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Various cellular components found within bovine compact morulae are illustrated in Figure 1. Examples of lipid droplets (L), vacuoles (V), mitochondria (M), and nuclei (N) within the cytoplasm, as well as intercellular space (IS) and microvilli (*), are depicted. Mature (MM) and vacuolated mitochondria (VM) are depicted in Figure 2, A and B. Immature (IM) and hooded mitochondria (HM), both of which were classified as immature mitochondria, are illustrated in Figure 2, C and D. Apoptotic bodies (AB) and inclusion bodies (IB) were also evaluated and are depicted in Figure 3.



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  		FIG. 1. General overview of cellular ultrastructure of a bovine compact morula (x5280); lipid droplets (L), vacuoles (V), mitochondria (M), nuclei (N), intercellular space (IS), and microvilli (*)



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  		FIG. 2. Mitochondrial types within bovine compact morulae. A) Mature mitochondria (MM, x24 000); B) vacuolated mitochondria (VM, x24 000); C) immature mitochondria (IM, x24 000); D) hooded mitochondria (HM, x24 000)



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  		FIG. 3. Inclusion bodies and apoptotic bodies (IB and AB, x8000) in a bovine compact morula

The volume density of lipid in compact morulae originating from the three in vitro culture treatments (IVPS, IVPSR, and mSOF) was increased (P < 0.05) compared with those produced in vivo (Fig. 4). Compact morulae produced in the IVPS treatment had an increased (P < 0.05) proportional volume of vacuoles compared with embryos produced either in vivo or in the IVPSR or mSOF treatments (Fig. 5).



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  		FIG. 4. Volume density of lipid for compact morulae from MO, IVPS, IVPSR, and mSOF treatments (a,b P < 0.05)



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  		FIG. 5. Volume density of cytoplasmic vacuoles for compact morulae from MO, IVPS, IVPSR, and mSOF treatments (a,b P < 0.05)

The volume density of mature mitochondria was decreased approximately 8-fold (P < 0.05) in compact morulae from IVPS, IVPSR, and mSOF compared with those produced in vivo (Table 1). Compact morulae from the IVPS medium had a reduced (P < 0.05) density of immature mitochondria compared with those from the IVPSR and mSOF treatments. There was no effect of treatment on the proportional volume of vacuolated mitochondria. Finally, the volume density of total mitochondria was decreased (P < 0.05) in compact morulae produced in the IVPS medium compared with that for compact morulae produced either in vivo or in the IVPSR or mSOF treatments (Table 1).


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  		TABLE 1. Effect of treatment on the volume density (%) of mitochondrial type in bovine compact morulae (least-squares means ± SEM)

The proportional volume of cytoplasm in compact morulae from the IVPS, IVPSR, and mSOF treatments was increased (P < 0.05) compared with that for embryos produced in vivo (Table 2). For morulae produced in the IVPS medium, the volume density of nuclei was significantly reduced compared with that for morulae produced in vivo (P < 0.05). The concurrent increase in volume density for cytoplasm and decrease in density for nuclei for compact morulae in the IVPS treatment resulted in a cytoplasmic-to-nuclear ratio that was significantly greater (P < 0.05) than that for compact morulae produced in vivo (Table 2). There was no effect of treatment on the volume densities for intercellular space, debris, inclusion bodies, or apoptotic bodies (Table 2).


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  		TABLE 2. Effect of treatment on the volume density (%) of cellular components in bovine compact morulae (least-squares means ± SEM)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although in vitro culture has been widely adopted as a method for the production of mammalian embryos, these systems can result in embryos with varying morphology compared with those produced in vivo [7, 9–11]. Ultrastructural characteristics of embryos from different production systems can be compared in an unbiased manner using morphometric methods. However, limited information is available in the literature concerning morphometric evaluation of mammalian embryos [41–43]. To our knowledge, the data reported in this paper represent the first application of morphometric techniques to quantify the effects of culture on ultrastructural characteristics of preimplantation bovine embryos.

The age of compact morulae produced in vitro for this study was based upon the interval postinsemination. In contrast, bovine embryos produced in vivo reach the compact morula-blastocyst transition between Days 6 and 8 postestrus [44] with the first appearance of early blastocyst stage embryos occurring around Day 7 [14]. Variance in the day of collection of compact morulae produced in vivo may result from several factors. First, because the donor cattle used in this study were observed for estrus at approximately 12-h intervals, detection of first standing estrus may not have been entirely coordinated with the actual time of true estrus initiation. Thus, embryos may have been deviated in development by up to 12 h (0.5 days) relative to their stated day of gestation. Second, although embryos from superovulated donors have been suggested to be representative of in vivo embryos obtained from nonstimulated animals [45], multiple follicles may ovulate over an extended period of time [15], contributing to variance in stage of development on the day of embryo collection. Finally, morulae produced in vivo remain in the compacted stage for more than one cleavage division and undergo the transition to blastocyst more slowly than those embryos produced in vitro [9]. Therefore, it is not surprising that compact morulae used in this study were recovered at both Day 6 and Day 8 postestrus.

An increase in lipid was observed in compact morulae produced in vitro compared with those produced in vivo. Interestingly, this increase occurred regardless of the composition of the medium in which the embryos were cultured. It has been suggested that an increased amount of lipid in embryos cultured in vitro results from uptake of lipid from serum in the culture medium [17, 18]. However, on the basis of data presented in this study, increases in lipid density occurred equally in compact morulae cultured in a completely serum-free medium and in serum-supplemented media. Therefore, the increased volume density of lipid in embryos cultured in vitro may result from membrane breakdown in response to a nonphysiological culture environment rather than uptake from the culture media. Alternatively, lipid may have accumulated as a result of insufficient metabolism by mitochondria present in compact morulae produced in vitro [19]. This latter mechanism would be consistent with the observed reduction in volume density of mature mitochondria for embryos produced in all three in vitro culture media. The sequestering of lipid substrates into the citric acid cycle occurs in the mitochondria [46]. In addition, the numerous folds of cristae within mature mitochondria contain the enzymes directly responsible for the production of ATP [47]. Therefore, a reduction in the volume density of mature mitochondria may contribute to the buildup of nonmetabolized lipid.

The volume density of total mitochondria was greater for embryos from the in vivo, IVPSR, and mSOF treatments than for those from the IVPS treatment. This resulted, in part, from the increased volume density of immature mitochondria in compact morulae produced in the serum-restricted and the serum-free treatments compared with those from the IVPS treatment. Immature mitochondria are organelles that have few, peripherally localized cristae. In primate embryos, this type of mitochondrion has been noted as typically found during the early cleavage and morula stages of development [22, 37]. Because immature mitochondria have fewer cristae, they may also have a decreased ability to metabolize lipid and produce ATP.

Cell death occurs as a normal part of in vivo embryo development [38]. In the primate, vacuolated mitochondria have been associated with the degeneration of aging oocytes and subsequent cell death [38]. In the present study, no differences were found in the volume density of vacuolated mitochondria for embryos from the four treatment groups. In addition, no differences were found among treatments in the volume densities of other organelles typically associated with cell degeneration including inclusion bodies, apoptotic bodies, or debris. Dead or dying cells have been found in otherwise normal primate preimplantation stage embryos produced in vivo [38]. Thus, it appears that production of compact morulae in vitro did not contribute to an increase in the occurrence of cell death compared with that found in embryos produced in vivo.

The presence of cytoplasmic vacuoles has been noted in primate [37, 39] and mouse embryos produced in vivo [48] as well as in bovine embryos produced in vitro [21, 23]. These vacuoles frequently contain cellular debris resulting from autophagy or ingestion of embryonic cell fragments [39]. It has been suggested that vacuoles are indicative of abnormal [38] or delayed developmental differentiation [21]. Because compact morulae produced in the IVPS culture treatment exhibited an increased volume density of vacuoles relative to embryos in the other treatment groups, the IVPS embryos may be more compromised in their development.

Compact morulae produced in the IVPS treatment also had an increased cytoplasmic-to-nuclear ratio compared to those produced in vivo. This resulted from both an increase in the volume density of cytoplasm and a decrease in the volume density of nuclei. The increased volume density of cytoplasm observed in morulae produced in vitro may result from incomplete compaction [8, 9]. Alternatively, the increased volume density of cytoplasm may have resulted from the occurrence of fewer cell divisions [6]. Compact morulae produced in vitro had fewer cells than those produced in vivo [9]. A decrease in the volume density of nuclei would be consistent with the presence of fewer blastomeres in compact morulae from the IVPS treatment.

Embryos produced in vitro have been reported to have a reduced degree of compaction [9] and cell-to-cell coupling compared to those produced in vivo [10]. If this were true, it might be expected that a greater volume density of intercellular space would have been present in embryos produced in vitro. However, in the present study, no differences were found in the volume densities for intercellular space in embryos from any of the four treatments. This discrepancy may be a result of differences in methods used for embryo production or for assessment of intercellular space [9, 10].

On the basis of a subjective evaluation of ultrastructural morphology, it was suggested that bovine embryos produced in serum-free culture medium have morphology comparable with that of embryos produced in vivo [24]. In contrast, based on morphometric analysis in the present study, the ultrastructure of embryos produced in serum-free culture (mSOF) was not comparable to that of embryos produced in vivo. These discordant conclusions may have resulted from differences in methods used for embryo production or for the evaluation of embryo ultrastructure [24].

Agreement among evaluators is reduced when determinating stage of development of compact morulae produced in vitro [12, 13]. Furthermore, pregnancy rates following transfer of compact morulae produced in vitro [14] were lower than those obtained after transfer of compact morulae produced in vivo [15]. Data presented in this study may provide some insights into the basis for the difficulties found when evaluating bovine compact morulae produced in vitro. Ultrastructural alterations resulting from in vitro culture could influence the assessment of both stage of development and quality grade of compact morulae. This, in turn, could have direct implications in resulting pregnancy rates.

In summary, on the basis of data in the present study, bovine embryos produced by in vitro methods were not comparable at an ultrastructural level to those produced in vivo. Overall, in vitro culture resulted in compact morulae with an increased amount of lipid and a decreased density of mature mitochondria. Compact morulae cultured entirely in serum-supplemented medium (IVPS) had the greatest degree of morphological deviation, with decreases in the densities of both mature and total mitochondria, an increased density of vacuoles, and an increased cytoplasmic-to-nuclear ratio.


   ACKNOWLEDGMENTS
 
The authors would like to acknowledge Brendalyn Bradley-Kerr for assistance in specimen preparation, identification of cellular components, and preparation of publication photographs.


   FOOTNOTES
 
First decision: 6 October 1999.

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

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

Accepted: January 4, 2000.

Received: September 8, 1999.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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