Msx2 Marks Spatially Restricted Populations of Mesenchymal Precursors.

Craniofacial development requires a set of patterning codes that define the identities of postmigratory mesenchymal cells in a region-specific manner, in which locally expressed morphogens, including fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs), provide instructive cues. Msx2, a bona fide target of BMP signaling, is a transcription factor regulating Runx2 and osterix (Osx), whose mutations are associated with cranial deformities in humans. Here we show that Msx2 defines osteo-chondro precursor cells in specific regions of the craniofacial mesenchyme at the postmigratory stage, particularly in the mandibular process and the posterior cranial vault. Analysis of Msx2-creER mice revealed that early mesenchymal cells in proximity to the BMP4-expressing mesenchyme were marked upon tamoxifen injection, and their descendants contributed to diverse types of mesenchymal cells in the later stage, such as chondrocytes and perichondrial cells of the transient cartilage, as well as osteoblasts and suture mesenchymal cells. By contrast, Osx-creER marked osteoblast precursors at the later stage, and their descendants continued to become osteoblasts well into the postnatal stage. Therefore, Msx2 marks spatially restricted populations of mesenchymal precursor cells with diverse differentiation potential, suggesting that extrinsic molecular cues can dictate the nature of postmigratory mesenchymal cells in craniofacial development.

Treatment with the MAPK kinase inhibitor U0126 during the first two hours of in vitro maturation improves bovine oocyte developmental competence.

This study examined the effects of treatment with U0126, which inhibits MAPK by inhibiting MAPK kinase, during the first 2 hr of in vitro maturation on bovine developmental competence and on gap junction (GAPJ) communication between the oocyte and cumulus cells. The percentage of oocytes developing to the blastocyst stage in the group treated with 5 µM U0126 (28%) was significantly higher than that in controls (15%, p < .05), while that in the group treated with 10 µM U0126 (18%) was not. Breakdown of the GAPJs was delayed in the group treated with 5 µM U0126 when compared to controls, as estimated by immunohistochemical examination of connexin 43, which is a primary constituent of the GAPJs. These results indicate that treatment with 5 µM U0126 during in vitro maturation delays GAPJ breakdown and improves bovine oocyte developmental competence.

Diverse contribution of Col2a1-expressing cells to the craniofacial skeletal cell lineages.

OBJECTIVES: Craniofacial skeletal development requires deliberate coordination of two distinct mechanisms of endochondral and intramembranous ossification. Col2a1-expressing cells encompass growth-associated skeletal progenitors in endochondral bones of the limb. The objective of this study was to determine the contribution of Col2a1-expressing cells to the craniofacial skeletal cell lineages. We hypothesize that Col2a1-expressing progenitors significantly contribute to various modes of ossification associated with the craniofacial development. METHODS: Cellular fates of Col2a1-expressing cells were studied based on a cre-loxP system using a Col2a1-cre transgene and an R26R-tdTomato reporter allele. We analysed three distinct locations of the craniofacial skeletal complex representing unique ossification mechanisms: the cranial base, the calvaria and the mandibular condyle. RESULTS: Col2a1-cre consistently marked a majority of skeletal cells in the cranial base. Interestingly, Col2a1-cre also marked a large number of osteoblasts and suture mesenchymal cells in the calvaria, in addition to chondrocytes in the underlying transient cartilage. In the mandibular condyle, Col2a1-cre marked chondrocytes and osteoblasts only during the growth phase. CONCLUSIONS: Col2a1 is expressed by progenitors of the skeletal lineage in canonical endochondral bone formation occurring in the cranial base. In contrast, other ossification mechanisms of the craniofacial complex utilize Col2a1-expressing cells in a different manner, whereby Col2a1 may be expressed in more differentiated or transient cell types of the skeletal lineage.

The relationship between oxygen consumption rate and viability of in vivo-derived pig embryos vitrified by the micro volume air cooling method.

The aim of this study was to assess the viability of vitrified-warmed in vivo-derived pig embryos after measuring the oxygen consumption rate. Six days after artificial insemination, blastocysts were collected from gilts and vitrified by the micro volume air cooling method. The oxygen consumption rate was measured in 60 vitrified-warmed embryos, which were then cultured for 48h to assess the viability. The survival (re-expansion) rate of embryos after warming was 85.0%. The average oxygen consumption rate of embryos immediately after warming was greater in embryos which could re-expand during subsequent culture (F=0.75±0.04) than that in those which failed to re-expand (F=0.33±0.05). Moreover, the oxygen consumption rate of vitrified-warmed embryos was greater in the hatched (F=0.88±0.06) than that in the not-hatched group (F=0.53±0.04). When the oxygen consumption rate of the vitrified-warmed embryos and the numbers of viable and dead cells in embryos were determined, there was a positive correlation between the oxygen consumption rate and the number of live cells (P<0.01, r=0.538). A total of 29 vitrified embryos after warming and measuring the oxygen consumption rate were surgically transferred into uterine horns of two recipients. Both of the recipients become pregnant and farrowed 12 healthy piglets. These results demonstrate that the oxygen consumption rate of vitrified-warmed pig embryos can be related to the number of live cells and that the measurement of oxygen consumption of embryos after cryopreservation may be useful for estimating embryo survivability.

Relationships between oxygen consumption rate, viability, and subsequent development of in vivo-derived porcine embryos.

Oxygen consumption rate of in vivo-derived porcine embryos was measured, and its value as an objective method for the assessment of embryo quality was evaluated. Embryos were surgically collected 5 or 6 days after artificial insemination (AI), and oxygen consumption rate of embryos was measured using an embryo respirometer. The average oxygen consumption rate (F × 10(14)/mol s(-1)) of the embryos that developed to the compacted morula stage on Day 5 (Day 0 = the day of artificial insemination) was 0.58 ± 0.03 (mean ± standard error of the mean). The Day-6 embryos had consumption rates of 0.56 ± 0.13, 0.87 ± 0.06, and 1.13 ± 0.07 at the early blastocyst, blastocyst, and expanded blastocyst stages, respectively, showing a gradual increase as the embryos developed. Just after collection, the average oxygen consumption rates of embryos that hatched and of those that did not hatch after culture were 0.60 ± 0.04 and 0.50 ± 0.04 for Day 5 (P = 0.08) and 1.05 ± 0.09 and 0.77 ± 0.05 for Day 6 (P < 0.05), respectively. The value and probability of discrimination by measuring the oxygen consumption rates of embryos to predict their hatching ability after culture were 0.56 and 63.6% for Day-5 embryos and 0.91 and 68.4% for Day-6 blastocysts, respectively. When Day-5 embryos were classified based on the oxygen consumption rate and then transferred non-surgically to recipient sows, three of the seven sows, to which embryos having a high oxygen consumption rate (≥ 0.59) were transferred, became pregnant and farrowed a total of 20 piglets. However, none of the four sows, to which embryos having low oxygen consumption rate (< 0.59) were transferred, became pregnant. These results suggest that the viability of in vivo-derived porcine embryos and subsequent development can be estimated by measuring the oxygen consumption rate.

Superovulatory response in Japanese Black cows receiving a single subcutaneous porcine follicle-stimulating hormone treatment or six intramuscular treatments over three days.

To reduce labor for superovulation treatment by twice-daily intramuscular (im) administration of FSH for more than 3 to 4 days, we investigated the superovulatory responses of Japanese Black cows to porcine FSH (pFSH) used as a single subcutaneous (sc) administration at two different doses in two different volumes of saline. In experiment 1, 20 Armour units (AU) of pFSH dissolved in either 10 mL (treatment A; n = 14) or 50 mL (treatment B; n = 14) of saline was administered subcutaneously in the neck region. In experiment 2, 30 AU of pFSH dissolved in either 10 mL (treatment C; n = 15) or 50 mL (treatment D; n = 15) of saline was administered subcutaneously in the neck region. The control animals in experiment 1 (n = 14) and experiment 2 (n = 15) received 20 AU of pFSH administered intramuscularly twice daily in decreasing doses for more than 3 days. In experiment 1, mean (±SEM) numbers of CL (15.4 ± 2.5, 18.1 ± 3.4, and 17.2 ± 2.6), total number of ova and embryos (12.9 ± 1.4, 15.9 ± 3.5, and 16.2 ± 2.8), and transferable embryos (7.5 ± 2.0, 10.4 ± 2.8, and 8.0 ± 2.1) did not differ among treatments A, B, and control. In experiment 2, mean (±SEM) numbers of CL (20.5 ± 4.3, 20.4 ± 2.7, and 20.1 ± 3.4), total number of ova and embryos (21.7 ± 4.2, 17.3 ± 3.4, and 16.5 ± 3.2), and transferable embryos (8.1 ± 1.6, 9.3 ± 2.2, and 9.5 ± 1.9) did not differ among treatments C, D, and control. Although there were no differences in serum pFSH concentrations among the three treatments at each of the time points in experiment 1, in experiment 2, the serum pFSH concentration at 6 and 8 hours after pFSH administration in treatment C (3.1 ± 0.8, 2.7 ± 0.5 ng/mL, mean ± SEM) was significantly greater (P < 0.05) than in the control (0.7 ± 0.1, 1.1 ± 0.2 ng/mL). At 10 hours after administration, the pFSH concentration had decreased and there were no differences among the three treatments at subsequent time points. These results suggest that increasing the volume of saline or the dose of pFSH does not affect the absorption pattern of pFSH administered as a single sc administration. In conclusions, single sc administration of pFSH at a dose of 20 or 30 AU dissolved in 10 or 50 mL of saline is able to induce a superovulatory response comparable with that obtained by twice-daily im administration in Japanese Black cows.

Colonization of denture plaque by respiratory pathogens in dependent elderly.

OBJECTIVES: Recently, there has been a resurgence of interest in the interactions between oral conditions and a number of prevalent systemic diseases. The morbidity and mortality of the dependent elderly that result from aspiration pneumonia have been recognized as a major geriatric health problem. The purpose of this study was to gain more information on the microflora of plaque on dentures and to assess the existence of oral infectious pathogens potentially causing the respiratory disease in the dependent elderly. SUBJECTS AND METHODS: The denture bacterial flora of 50 dependent elderly were examined to identify microorganisms by the culture method. RESULTS: 18 species of microorganisms were detected in denture plaque in this study. A variety of pathogens with the potential to cause respiratory infection pathogens colonized on the dentures of dependent elderly. CONCLUSION: The results of the present study revealed that bacteria that commonly cause respiratory infection colonized on the dentures of dependent elderly, suggesting that denture plaque may function as a reservoir of potential respiratory pathogens to facilitate colonization on the oropharynx.

Sodium fluoride increases intracellular calcium in rat renal epithelial cell line NRK-52E.

In our previous experiment using rats, fluoride was reported to cause renal calcification, whose mechanism was deduced to be due to an increase in parathyroid hormone (PTH) secretion. However fluoride-induced renal calcification that was independent of PTH has not been understood well in the nephron of fluoride-treated animals. Thus, we examined the effect of sodium fluoride on intracellular calcium mobilization in a normal rat kidney epithelial cell line (NRK-52E cells). The calcium accumulation was found to be remarkably increased by the addition of sodium fluoride (NaF). The elevation of [Ca2+]i was demonstrated to be due to calcium entry through nifedipine-sensitive calcium channels. In addition, fluoride activates phospholipase C, but inositol 1,4,5-triphosphate (IP3) didn't induce Ca2+ release from the endoplasmic reticulum (ER). Moreover, fluoride alone was deduced to enhance the activity of ER-type Ca2+-ATPase. Finally, on the mechanism of fluoride-induced calcium accumulation in NRK-52E cells, fluoride may activate phospholipase C to generate IP3 and diacylglycerol, and these increases can be elucidated to induce calcium entry through dihydropiridine-sensitive calcium channels. Moreover, fluoride was found to stimulate calcium accumulation through ER-type Ca2+-ATPase into the endoplasmic reticulum. The elevation of ER-type Ca2+-ATPase activity by fluoride was elucidated to operate as a regulatory system to protect against abnormally higher increases in cytosolic calcium concentration via an increase of calcium influx into the endoplasmic reticulum.

Effect of megestrol acetate or melengestrol acetate on preneoplastic and neoplastic mammary growth in mice.

Pellets of megestrol acetate and melengestrol acetate were implanted subcutaneously every one and two months, respectively, into 3-5 month old SHN virgin mice. Spontaneous mammary tumourigenesis was significantly enhanced by both steroids. The formation of preneoplastic mammary hyperplastic alveolar nodules (HAN) was, in contrast, inhibited by both progestins. As these results closely resemble the effects of progesterone and of medroxyprogesterone acetate on the development of mammary tumours both in the SHN mouse and in the woman, it seems likely that this animal model may prove valuable for the study of human breast cancer that is no longer responsive to progestins.

Medroxyprogesterone acetate enhances spontaneous mammary tumorigenesis and uterine adenomyosis in mice.

Based on our previous findings that medroxyprogesterone acetate (MPA) significantly suppressed the formation of preneoplastic mammary hyperplastic alveolar nodules in mice, and that this suppression persisted for some time, we studied the effects of different schedules of MPA treatment on spontaneous mammary tumorigenesis and uterine adenomyosis in SHN virgin mice. Mice received a subcutaneous pellet of MPA every 2 months: I) during the limited period of 1-3 months of age; II) throughout the experiment beginning at 6-8 months of age; and III) throughout the experiment beginning at 2-3 months of age. All treatments significantly enhanced mammary tumorigenesis with little difference in the effects among treatments. The progression of uterine adenomyosis was also stimulated in Experiments I and III, but not in Experiment II. These results are in good accord with our previous observations with progesterone, indicating that MPA has progesterone-like effects on mammary and uterine lesions of mice.

Counteraction by combined treatment with high dose of oestrogen and somatostatin of mammary growth suppression in mice.

The interaction between somatostatin (SMS) and high doses of oestrogen on mammary gland growth was examined in C3H/He virgin mice. Mammary gland growth was significantly suppressed by the subcutaneous implantation of pellets of oestradiol benzoate diluted to 1/1000 or 1/500 or by injection twice daily of 50ng SMS 201-995 between 25 and 55 days of age. However, the mammary gland growth of mice receiving SMS and oestrogen in combination was markedly stimulated compared to that of mice given the respective agents. These results indicate that the inhibitory effects of somatostatin and oestrogen on mammary growth were apparently counteracted by the treatment in combination.

Effect of chronic suppression of growth hormone secretion, by SMS 201-995 on lactation in mice.

The effect of chronic suppression of growth hormone (GH) secretion by SMS 201-995 on lactation was studied in primiparous C3H/He mice. Mammary gland DNA content on day 12 of lactation was significantly lower in SMS 201-995 treated mice than in the control. There were little differences between groups in mammary gland RNA content and litter growth on day 12 of lactation. That was associated with a slightly higher RNA/DNA ratio and a significant increase in food intake during lactation. These results indicate that inhibited mammary gland growth by GH suppression has little effect on lactation. The smaller mammary gland can compensate by increasing its secretory activity.