Category Archives: Morphometry

Ultrastructural Morphometry of Bovine Compact Morulae Produced In Vivo or In Vitro: DISCUSSION(5)

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. 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.

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Ultrastructural Morphometry of Bovine Compact Morulae Produced In Vivo or In Vitro: DISCUSSION(4)

DISCUSSION(4)

The presence of cytoplasmic vacuoles has been noted in primate and mouse embryos produced in vivo as well as in bovine embryos produced in vitro. These vacuoles frequently contain cellular debris resulting from autophagy or ingestion of embryonic cell fragments. It has been suggested that vacuoles are indicative of abnormal or delayed developmental differentiation. 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.

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Ultrastructural Morphometry of Bovine Compact Morulae Produced In Vivo or In Vitro: DISCUSSION(3)

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. Because immature mitochondria have fewer cristae, they may also have a decreased ability to metabolize lipid and produce ATP.

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Ultrastructural Morphometry of Bovine Compact Morulae Produced In Vivo or In Vitro: DISCUSSION(2)

DISCUSSION(2)

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. 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.

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Ultrastructural Morphometry of Bovine Compact Morulae Produced In Vivo or In Vitro: DISCUSSION(1)

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. Ultra-structural 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. 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.

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Ultrastructural Morphometry of Bovine Compact Morulae Produced In Vivo or In Vitro: RESULTS

RESULTS

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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Ultrastructural Morphometry of Bovine Compact Morulae Produced In Vivo or In Vitro: MATERIALS AND METHODS(3)

Electron Microscopy

Embedment procedures were modified from Dykstra. 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. 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. 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 ^m2 (mean ± SEM) was analyzed for each compact morula.

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Ultrastructural Morphometry of Bovine Compact Morulae Produced In Vivo or In Vitro: MATERIALS AND METHODS(2)

MATERIALS AND METHODS(2)

In Vitro Embryo Production

Cumulus-oocyte complexes (COC) were aspirated, matured, and fertilized in vitro as described by Farin and Farin. Briefly, ovaries from Holstein cattle were collected at a local abattoir and held in saline with 0.75 ^g/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; ). 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 ^g/ml LH, 5 ^g/ml FSH, 1 ^g/ml estradiol, 200 ^M pyruvate sodium salt, and 50 ^g/ml gentamicin. Cultures were maintained in an atmosphere of 5% CO2 in air with 100% humidity.

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Ultrastructural Morphometry of Bovine Compact Morulae Produced In Vivo or In Vitro: MATERIALS AND METHODS(1)

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 F2a was obtained from Phar-macia & Upjohn (Lutalyse; Kalamazoo, MI). Folltropin was obtained from Vetrepharm Canada (London, ON, Canada).

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Ultrastructural Morphometry of Bovine Compact Morulae Produced In Vivo or In Vitro: INTRODUCTION

INTRODUCTION

Compaction of morula-stage mammalian embryos is required for proper blastocyst formation and leads to differentiation of the inner cell mass and trophectoderm. Many structural events are associated with compaction, including blastomere polarization, blastomere flattening, and gap junction formation. In bovine and human embryos, the events of compaction are dependent on embryonic age, blastomere number, and number of cell divisions.

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