Matrix Gla Protein Is a Developmental Regulator of Chondrocyte Mineralization And, When Constitutively Expressed, Blocks Endochondral and Intramembranous Ossification in the Limb

Matrix GLA protein (MGP), a γ-carboxyglutamic acid (GLA)–rich, vitamin K–dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage–dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a γ-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

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282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab282 ◽

2006 ◽

Vol 18 (2) ◽

pp. 248

Author(s):

S.-G. Lee ◽

C.-H. Park ◽

D.-H. Choi ◽

H.-Y. Son ◽

C.-K. Lee

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Transgenic Animals ◽

Cell Number ◽

Blastocyst Stage ◽

Cell Stage ◽

Vitro Fertilization

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

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81 HIGH THROUGHPUT VITRIFICATION OF IN VIVO FERTILIZED AND IN VITRO CULTURED PORCINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab81 ◽

2011 ◽

Vol 23 (1) ◽

pp. 146

Author(s):

C. N. Murphy ◽

L. D. Spate ◽

B. K. Bauer ◽

R. S. Prather

Keyword(s):

Ethylene Glycol ◽

Uv Light ◽

Cell Number ◽

Total Cell ◽

Swine Industry ◽

Total Cell Number ◽

Cell Stage ◽

High Osmolarity

One barrier to successfully making embryo transfer viable in the swine industry is an inability to consistently cryopreserve oocytes and embryos. This process is made difficult by the high lipid content of porcine oocytes and embryos. The objective of this study was to test the in vivo fertilized embryo’s sensitivity to vitrification. Gilts were inseminated on the first day of standing oestrus (Day 0) and then again 12 h later. On Day 2 the oviducts and tip of the uterine horns were flushed with PVA-treated TL-HEPES and 2-cell stage embryos were collected and placed into PVA-treated TL-HEPES and centrifuged at 17 000 × g. The treatment groups were 1) 300 mOsmo centrifuged for 6 min, 2) 500 mOsmo centrifuged for 6 min, 3) 500 mOsmo centrifuged for 12 min, and 4) 500 mOsmo centrifuged for 18 min. After centrifugation the embryos were transferred to Porcine Zygote Medium 3 (PZM3) and cultured to Day 6 or 7 at which point blastocysts were vitrified using 10% DMSO, 10% ethylene glycol in M199 supplemented with 20% FBS (holding medium) for 2 min. Embryos were transferred to holding media with 20% DMSO and 20% ethylene glycol and drawn into an open pulled straw via capillary reaction; it was then submerged into LN2. Embryos were thawed using a step down concentration of 0.33 mM and then 0.2 mM sucrose in holding media each for 6–7 min and then were moved to holding medium alone for 6 to 7 min. The embryos were washed in PZM3, then transferred to 500 μL of PZM3 and cultured for 18 h. Re-expanded embryos were observed, and the nuclei of all embryos were stained with Biz-benzimide and visualised with UV light to determine total cell number. After the embryos were centrifuged and cultured, there was no difference in development to blastocyst (SAS Institute, Cary, NC, USA; Proc GLM) with a mean percentage blastocyst of 85.1% and an N of 54, 51, 53, and 51, respectively, for each treatment. After thawing, percentage of embryos re-expanded was 23.5a, 26.4a,b, 43.2a,b, and 45.6b, respectively. Data was analysed using a PROC GLM in SAS (P < 0.05), with 37, 43, 30, and 36 embryos in each group, respectively. No difference in total cell number across treatments was detected after analysis using PROC GLM in SAS (P < 0.05) with a mean cell number of 29.0. These data suggest that in vivo matured and fertilized blastocysts can survive high osmolarity treatment, centrifugation, and vitrification. The data also show that a high osmolarity treatment centrifuged for 18 min leads to a greater number of re-expanded embryos post-thaw, which may be attributed to better separation of the lipid. Funded by the NIH NCRR R21RR025879 and Food for the 21st Century.

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Development of Preimplantation Mouse Embryos in vivo and in vitro

Australian Journal of Biological Sciences ◽

10.1071/bi9820187 ◽

1982 ◽

Vol 35 (2) ◽

pp. 187 ◽

Cited By ~ 81

Author(s):

GM Harlow ◽

P Quinn

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Cell Number ◽

Blastocyst Stage ◽

Culture Conditions ◽

Cell Stage ◽

Preimplantation Mouse ◽

Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

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25 IN VITRO DEVELOPMENT OF AGGREGATED NUCLEAR TRANSFERRED EMBRYOS DERIVED FROM BOVINE CUMULUS CELLS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab25 ◽

2005 ◽

Vol 17 (2) ◽

pp. 162

Author(s):

S. Akagi ◽

B. Tsuneishi ◽

S. Watanabe ◽

S. Takahashi

Keyword(s):

Zona Pellucida ◽

Cumulus Cells ◽

Cell Number ◽

Blastocyst Stage ◽

Cell Stage ◽

In Vitro Development ◽

Functional Peptide ◽

Vitro Development

It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304–5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3–5 were used following culture in serum-starved medium for 5–7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264–272). NT embryos were cultured in a serum-free medium (IVD-101, Research Institute of Functional Peptide Co., Ltd., Shimojo, Yamagat, Japan). Eight-cell-stage embryos on Day 2 or 16- to 32-cell-stage embryos on day 4 were used for embryo aggregation after removal of the zona pellucida. A small depression was made in a 25-μL drop of TCM-199 with 50 μg/mL phytohemagglutinin (TCM199/PHA) or IVD-101 using a darning needle. Two or three NT embryos were placed into the depression in the drop of TCM199/PHA for 20 min. NT aggregates were then moved into the depression in the drop of IVD-101 and cultured until Day 7. In vitro development of NT aggregates was summarized in Table 1. There were no differences in the cell number and ICM ratio of blastocysts between non-aggregated zona-intact and zona-free embryos. All aggregates of three NT embryos developed to the blastocyst stage and the cell number of these blastocysts was significantly higher than that of non-aggregated NT blastocysts. These results indicate that removal of the zona pellucida does not affect the cell number and ICM ratio of blastocysts and that aggregates of three NT embryos can develop to blastocysts with high cell numbers which are equivalent to in vivo-derived embryos (166 ± 11, Knijn HM et al. 2003 Biol. Reprod. 69, 1371–1378). Table 1. Development, cell number, and ICM ratio of NT aggregates

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A contractile acto-myosin belt promotes growth anisotropy during the early stages of pectoral fin development in zebrafish

10.1101/512038 ◽

2019 ◽

Author(s):

Elena Kardash ◽

Hanh Nguyen ◽

Martin Behrndt ◽

Carl-Philipp Heisenberg ◽

Nadine Peyriéras ◽

...

Keyword(s):

Cell Number ◽

Limb Bud ◽

Central Position ◽

Anisotropic Growth ◽

Pectoral Fin ◽

Protein Activity ◽

Normal Shape ◽

Changes Over Time ◽

Fin Shape

SummaryThe zebrafish pectoral fin is an in vivo model for vertebrate limb formation, well suited to investigate the integration of molecular and cellular dynamics, the results of which translate into shaping the limb bud. We used the ratio between the lengths of the anterior-posterior (AP) and dorso-ventral (DV) axes as the descriptor of how fin shape changes over time. We showed that fin shape transitions from close to hemi-spherical (ratio 1. 36 ± 0.11) to semi-ellipsoid (ratio 1.64 ± 0.04) between 33 and 46 hours post fertilization (hpf). This shape transition coincided with the formation of a contractile “actin belt” at the distal rim of the fin bud along its AP axis. The actin belt emerged from a central position and expanded on both sides along the distal rim of the fin, thus marking the DV boundary between two rows of ectodermal cells. Formation of the actin belt depended on Rac protein activity, as suggested by FRET measurements using a Rac biosensor. 3D+time imaging of the developing fin in Rac-deficient embryos showed that anisotropic growth of the fin depends on the actin belt. Indeed, actin belt formation was dramatically reduced or even absent in the embryos without proper Rac activity. This correlated with isotropic growth of the fin bud from normal shape at 33 hpf to quasi hemispherical shape with AP/DV ratio ~1 13 hours later, without affecting cell number and overall bud volume. We propose that the formation of a contractile acto-myosin belt is essential to drive the pectoral fin’s early anisotropic growth.

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An analysis of the condensation process during chondrogenesis in the embryonic chick hind limb

Development ◽

10.1242/dev.33.3.581 ◽

1975 ◽

Vol 33 (3) ◽

pp. 581-606

Author(s):

P. V. Thorogood ◽

J. R. Hinchliffe

Keyword(s):

Hind Limb ◽

Mesenchymal Cell ◽

Cell Number ◽

Limb Bud ◽

Condensation Process ◽

Embryonic Chick ◽

Cellular Events ◽

Cell Counts

An analysis has been made of the pre-cartilaginous condensation stage in the development of the femur and tibia/fibula skeletal blastemata of the embryonic chick hind limb. Light microscopy serial sections were used to ‘map’ the mesenchymal cell condensations of both myogenic and chondrogenic anlagen in the limb-bud from stages 22 to 26 (Hamburger & Hamilton, 1951). Cell counts reveal that an increase in mesenchymal cell number per unit area occurs in the central chondrogenic locus at stage 24 (4½ days) prior to matrix formation. Electron microscopy, using a simultaneous double fixation with osmium and glutaraldehyde, reveals that the pre-chondrogenic cells are characterized by large areas of close surface contact between adjacent cells, as compared with the extensive intercellular spaces associated with undifferentiated mesenchymal cells. The results are discussed and related to other investigations of in vivo chondrogenesis and to analyses of cellular events during in vitro chondrogenesis. These observations are consistent with the theory that condensations are formed by a process of aggregation rather than by localized increased mitosis.

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Regulation of embryonic size in early mouse development in vitro culture system

Zygote ◽

10.1017/s0967199412000652 ◽

2013 ◽

Vol 22 (3) ◽

pp. 340-347 ◽

Cited By ~ 2

Author(s):

Tomoka Hisaki ◽

Ikuma Kawai ◽

Koji Sugiura ◽

Kunihiko Naito ◽

Kiyoshi Kano

Keyword(s):

Culture System ◽

Cell Number ◽

Mouse Development ◽

Cell Stage ◽

Relative Growth ◽

Development In Vitro ◽

Early Mouse ◽

Post Implantation

SummaryMammals self-regulate their body size throughout development. In the uterus, embryos are properly regulated to be a specific size at birth. Previously, size and cell number in aggregated embryos, which were made from two or more morulae, and half embryos, which were halved at the 2-cell stage, have been analysed in vivo in preimplantation and post-implantation development in mice. Here, we examined whether or not the mouse embryo has the capacity to self-regulate growth using an in vitro culture system. To elucidate embryonic histology, cells were counted in aggregated or half embryos in comparison with control embryos. Both double- and triple-aggregated embryos contained more cells than did control embryos during all culture periods, and the relative growth ratios showed no growth inhibition in an in vitro culture system. Meanwhile, half embryos contained fewer cells than control embryos, but the number grew throughout the culture period. Our data suggest that the growth of aggregated embryos is not affected and continues in an in vitro culture system. On the other hand, the growth of half embryos accelerates and continues in an in vitro culture system. This situation, in turn, implied that post-implantation mouse embryos might have some potential to regulate their own growth and size as seen by using an in vitro culture system without uterus factors. In conclusion, our results indicated that embryos have some ways in which to regulate their own size in mouse early development.

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32 EFFICIENCY OF FEMALE-DERIVED DONOR CELLS ON HIGH POSTNATAL SURVIVAL IN PIG CLONING

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab32 ◽

2005 ◽

Vol 17 (2) ◽

pp. 166

Author(s):

S.K. Cho ◽

M.R. Park ◽

D.N. Kwon ◽

E.K. Lee ◽

S.J. Kang ◽

...

Keyword(s):

Adenosine Monophosphate ◽

Cell Number ◽

Developmental Competence ◽

Cell Stage ◽

Male And Female ◽

Electric Pulses ◽

Donor Cells ◽

Fetal Fibroblasts

The present study was conducted to investigate the developmental competence of male and female somatic cell derived nuclear transfer (NT) porcine embryos and also the production and survival efficiency of cloned male and female piglets. Maturation of porcine COCs was accomplished by incubation in NCSU-23 medium supplemented with 0.6 mM cysteine, 10% porcine follicular fluid, 1 mM dibutyryl cyclic adenosine monophosphate, and 0.1 IU/mL human menopausal gonadotrophin for 20 h and then culture without dbcAMP and hMG for another 18 to 24 h. Fetal cells were isolated from a male fetus and two female fetuses, and cultured in ES-DMEM medium containing 10% FCS. Enucleated oocytes were fused with fetal fibroblasts (passage 4 to 15). Reconstructed embryos were cultured in NCSU-23 with 4 mg/mL BSA under mineral oil at 39°C in 5% CO2 in air for up to 6 days. NT eggs that had been activated with electric pulses and cultured for 1 or 2 days were transported to the experimental station in modified NCSU-23 with antibiotics. NT embryos were surgically transferred into the oviducts of recipients between Day 27 and Day 30; pregnancy was determined by ultrasound. The potential of NT embryos to develop into blastocysts was not different among donor cells of different origins. However, the mean cell number of in vivo female and male blastocysts (83.8 ± 46.2 to 99.2 ± 55.7) was higher than in in vitro culture of NT groups (31.4 ± 8.29 to 33.2 ± 10.15). A total of 11,535 NT embryos (1- to 8-cell stage) were surgically transferred into 66 surrogate gilts. Among fourteen pregnant gilts, four recipients aborted during the period of conception. Five pregnant gilts delivered fifteen female piglets, 1.28 ± 0.33 kg (0.48∼1.83 kg) in female piglets and 0.84±0.25 kg (0.45∼1.25 kg) in male piglets. Nine live cloned female (60.0%) and four male piglets (18.2%) were produced. According to these results, survival rates and birth weights of female cloned piglets were higher than those of cloned male piglets (P < 0.05). This study suggests that use of female, compared with male, fetal fibroblast cells as nuclear donors may increase cloning outcomes. This work was supported in part by a grant program from RDA(Biogreen21) and Cho-A, Republic of Korea.

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97 CLONED CALF PRODUCTION BY AGGREGATION OF SOMATIC CELL NUCLEAR-TRANSFERRED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab97 ◽

2007 ◽

Vol 19 (1) ◽

pp. 166

Author(s):

D. Yamaguchi ◽

W. Suzuki ◽

N. Adachi ◽

S. Akagi ◽

S. Watanabe ◽

...

Keyword(s):

Somatic Cell ◽

High Frequency ◽

Cell Number ◽

Blastocyst Stage ◽

Fibroblast Cells ◽

Cell Cloning ◽

Cell Stage ◽

Cell Numbers ◽

Donor Cells

A low pregnancy rate and high frequency of abortion are observed in bovine somatic cell cloning. It is suggested that one of the factors is the low cell number of nuclear-transferred (NT) embryos at the blastocyst stage compared with that of in vivo-derived embryos. We reported that aggregation of bovine NT embryos can develop to blastocysts with cell numbers that are equivalent to in vivo-derived embryos (Akagi et al. 2005 Reprod. Fertil. Dev. 17, 162 abst). In this study, we examined the in vivo development of aggregates of NT embryos after embryo transfer (ET). Following culture in serum-starved medium for 5 to 7 days, fibroblast cells were used as donor cells for NT. NT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Eight-cell stage embryos on Day 2 or 16- to 32-cell stage embryos on Day 4 were used for embryo aggregation after removal of the zona pellucida. Three NT embryos were placed into the depression in a drop of TCM-199 with 50 µg mL−1 phytohemagglutinin for 20 min, and NT aggregates were then moved into the depression in a drop of IVD-101 (Akagi et al. 2005). On Day 7 after NT, ET was performed nonsurgically. Results are summarized in Table 1. Pregnancy rates obtained with the aggregated NT embryos tended to be high compared with the single NT embryos. All pregnant cows in the single NT embryos and 6 of 8 pregnant cows in the aggregated NT embryos were aborted within 90 days. One cow carrying an aggregate of 3 Day 2 NT embryos went to term, and one carrying an aggregate of 3 Day 4 NT embryos is still pregnant at more than 7 months. These results demonstrate that aggregates of 3 NT embryos can develop to term in cattle. Table 1.In vivo development of aggregated NT embryos

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Actions of Hedgehog Proteins on Skeletal Cells

Critical Reviews in Oral Biology & Medicine ◽

10.1177/10454411990100040401 ◽

1999 ◽

Vol 10 (4) ◽

pp. 477-486 ◽

Cited By ~ 21

Author(s):

M. Iwamoto ◽

M. Enomoto-Iwamoto ◽

K. Kurisu

Keyword(s):

Bone Formation ◽

Cell Line ◽

Ectopic Expression ◽

Limb Bud ◽

Amino Terminal ◽

Chondrocyte Maturation ◽

Skeletal Formation ◽

Hedgehog Proteins

Recent advances in developmental and molecular biology during embryogenesis and organogenesis have provided new insights into the mechanism of bone formation. Members of the hedgehog gene family were initially characterized as patterning factors in embryonic development, but recently they have been shown to regulate skeletal formation in vertebrates. The amino terminal fragment of Sonic hedgehog (Shh-N), which is an active domain of Shh, has the ability to induce ectopic cartilage and bone formation in vivo. Shh-N stimulates chondrogenic differentiation in cultures of chondrogenic cell line cells in vitro and inhibits chondrogenesis in primary limb bud cells. These findings suggest that the regulation of chondrogenesis by hedgehog proteins depends on the cell populations being studied. Indian hedgehog (Ihh) is prominently expressed in developing cartilage. Ectopic expression of Ihh decreases type X collagen expression and induces the up-regulation of parathyroid hormone-related peptide (PTHrp) gene expression in perichondrium cells. A negative feedback loop consisting of Ihh and PTHrp, induced by Ihh, appears to regulate the rate of chondrocyte maturation. The direct actions of Shh and Ihh on stimulation of osteoblast differentiation are evidenced by the findings that these factors stimulate alkaline phosphatase activity in cultures of pluripotent mesenchymal cell line cells and osteoblastic cells and that these cells express putative receptors of hedgehog proteins. In conclusion, hedgehog proteins seem to be significantly involved in skeletal formation through multiple actions on chondrogenic mesenchymal cells, chondrocytes, and osteogenic cells.

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