Biomedical subjects
J W Pollard
Publications and source records attributed to J W Pollard.
Effect of ambient temperatures during oocyte recovery on in vitro production of bovine embryos.
Recovery of oocytes from ovaries collected at slaughter was carried out at three ambient temperatures (25 degrees, 30 degrees and 35 degrees C) to assess the effect on subsequent embryonic production in vitro. Oocytes recovered at each temperature were thereafter maintained at temperatures > or =35 degrees C as they were subjected to in vitro maturation, fertilization and culture (IVM/IVF/IVC). The oocytes and resulting embryos within each temperature group were subsequently evaluated for their rates of fertilization, cleavage and development to blastocysts, as well as for the number of cells/blastocyst. The results demonstrate that exposure of cumulus-ocyte-complexes (COCs) to temperatures below 35 degrees C during oocyte recovery is detrimental to optimal embryo production. Although the fertilization and cleavage rates of oocytes recovered at temperatures below 35 degrees C were not significantly lower than that of the controls, the percentage of oocytes recovered at 35 degrees C that developed to the blastocyst stage following fertilization and culture (33.7%) was significantly greater than those from oocytes recovered at either 25 degrees C (22.4%) or 30 degrees C (19.5%). The mean numbers of blastomeres/embryo were significantly lower in embryos derived from oocytes collected at either 25 degrees or 30 degrees compared with those collected at 35 degrees C. The results of this study suggest that exposure of COCs to temperatures below 35 degrees C during oocyte recovery may significantly decrease both the quantity and quality of embryos produced by in vitro methods.
Meiotic pachytene arrest in MLH1-deficient mice.
Germ line mutations in DNA mismatch repair genes including MLH1 cause hereditary nonpolyposis colon cancer. To understand the role of MLH1 in normal growth and development, we generated mice that have a null mutation of this gene. Mice homozygous for this mutation show a replication error phenotype, and extracts of these cells are deficient in mismatch repair activity. Homozygous mutant males show normal mating behavior but have no detectable mature sperm. Examination of meiosis in these males reveals that the cells enter meiotic prophase and arrest at pachytene. Homozygous mutant females have normal estrous cycles and reproductive and mating behavior but are infertile. The phenotypes of the mlh1 mutant mice are distinct from those deficient in msh2 and pms2. The different phenotypes of the three types of mutant mice suggest that these three genes may have independent functions in mammalian meiosis.
Leukemia inhibitory factor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation.
The uterine expression of leukemia inhibitory factor (LIF) is essential for embryo implantation in the mouse. Here, we describe the expression of LIF, related members of this group of cytokines, oncostatin M and ciliary neurotrophic factor, and the LIF receptor beta and glycoprotein gp130 in normal human tissues and in the endometrium of fertile women. Our results show that LIF is the only one of these factors expressed at detectable levels in the endometrium of women of proven fertility. LIF expression is restricted to the endometrial glands during the secretory/postovulatory phase but is not present in the endometrium during the proliferative/preovulatory phase. The LIF receptor beta is expressed during the proliferative and secretory phases of the cycle and is restricted to the luminal epithelium. The associated signal-transducing component of the LIF receptor, gp130, is also expressed in both the luminal and glandular epithelium throughout the cycle. These results suggest that uterine expression of LIF in humans, like mice, may have a role in regulating embryo implantation, possibly through an autocrine/paracrine interaction between LIF and its receptor at the luminal epithelium.
Effect of chilling bovine oocytes on their developmental competence.
Bovine oocytes are damaged when chilled to temperatures near 0 degree C. We have determined the temperatures at which this injury occurs, as well as its kinetics and the functional consequences for oocytes both at the germinal vesicle-stage (GV) and after in vitro maturation (IVM). Cooling GV oocytes had no effect on their nuclear maturation or fertilization. Compared to control oocytes held at 30 degrees C, the development of GV oocytes into blastocysts following maturation and fertilization was unaffected by cooling them to 20 degrees C for 30 min (blastocyst formation: 25% vs 26%, respectively), but development decreased after cooling them to 10 degrees C and 0 degree C (blastocyst: 6% and 1%, respectively). Cooling oocytes after maturation gave similar results, with no difference between controls and oocytes cooled to 20 degrees C (blastocyst: 25% and 26%, respectively). However, cooling them to 10 degrees C and 0 degree C did reduce development (blastocyst: 8% and 3%, respectively). Chilling oocytes to 0 degree C for 30 sec reduced their cleavage and blastocyst formation by > 40%; there was a high negative correlation between the length of exposure and subsequent survival, both for GV-stage and for IVM oocytes. The extreme sensitivity of both GV and IVM oocytes to chilling can explain the limited success obtained for cryopreservation of bovine oocytes by conventional slow-cooling procedures.
Absence of colony-stimulating factor-1 in osteopetrotic (csfmop/csfmop) mice results in male fertility defects.
Previous studies have shown that the mononuclear phagocyte growth factor, colony-stimulating factor-1 (CSF-1), has an important role in female reproduction. Mating experiments with osteopetrotic (csfmop/csfmop) mice, which possess an inactivating mutation in the CSF-1 gene, suggested that there are male, as well as female, reproductive defects. In the present study, we have shown that male csfmop/csfmop mice have a sevenfold lower concentration of circulating testosterone (T) and a significantly lower intratesticular T concentration than wild-type mice. These lowered T concentrations were associated with a reduction in mating capability and a reduction in the number of viable sperm. Reconstitution of male csfmop/csfmop mice with either circulating T in the adult or circulating CSF-1 throughout the postnatal period completely restored viable sperm numbers and significantly restored sexual behavior. These observations, coupled with the close association of Leydig cells with testicular macrophages and the proposed function of these macrophages in the regulation of Leydig cell steroidogenesis, suggest that CSF-1-regulated testicular macrophages play an important role in male reproduction.
CSF-1 deficiency in mice results in abnormal brain development.
Colony stimulating factor-1 (CSF-1) was initially identified as a growth factor for mononuclear phagocytes. This study examines the role of CSF-1 in the development of the central nervous system (CNS). CSF-1 treatment of neurons cultured from embryonic brain promoted survival and process outgrowth in a dose-dependent manner. By contrast, CSF-1 treatment of neurons cultured from the osteopetrotic (op/op) mouse, a null mutant for CSF-1, promoted significantly less process outgrowth, suggesting that there are neural abnormalities in op/op animals. Nuclease protection assays were used to determine whether CSF-1 and its receptor are expressed at times appropriate to regulate neural development. Both CSF-1 and its receptor are expressed in developing mouse brain, with a unique pattern of CSF-1 mRNA splice variant expression encoding secreted, and not membrane-bound, growth factor. To determine whether brain function is altered by null mutation of CSF-1, op/op mice were examined using electrophysiologic assays. Brainstem auditory and visual evoked potentials were both abnormal in op/op mice. Further, intracortical recordings revealed aberrant neuronal function within visual cortex and alterations in the cortical circuitry that balances excitation and inhibition. Daily CSF-1 injection of postnatal op/op mice largely rescued the abnormal neural phenotype, confirming that the absence of CSF-1 during development is responsible for the abnormalities. The effects of CSF-1 on cultured embryonic neural cells, the developmentally appropriate expression of CSF-1 and its receptor, and the neurological abnormalities in op/op mice suggest a role for CSF-1 in brain development.
Direct observation and quantification of macrophage chemoattraction to the growth factor CSF-1.
The cloned mouse macrophage cell line, BAC1.25F, resembles primary macrophages in its dependence on colony stimulating factor-1 (CSF-1) for both viability and proliferation. Re-addition of CSF-1 to cytokine-deprived cells, which are rounded with diffusely organised F-actin, stimulates rapid cell spreading and cell polarisation. Using the Dunn chemotaxis chamber the movement of stimulated macrophages was monitored over a 2 hour period. Cells restimulated with 1.32 nM human recombinant CSF-1 migrated at a mean rate of 7.71 microns per hour, but showed no directional preferences. In a linear concentration gradient of CSF-1, cytokine-deprived cells were again stimulated to migrate and the mean rate of cell motility, at 6.88 microns per hour, was not significantly different from that measured in an isotropic environment of CSF-1. However, there was a strong preference for the cells to orientate so that their long axes aligned with the CSF-1 gradient and they migrated preferentially towards the source of CSF-1. Migrating cells contained abundant F-actin within the leading lamellae as judged by confocal imaging of fluorescent phalloidin, but the actin was not arranged into stress fibre-like structures. These data support the proposition that CSF-1 is both a chemokinetic and chemotactic agent for macrophages. Tumour necrosis factor (TNF-alpha) failed to stimulate cell migration and thus was neither chemokinetic nor a chemotactic agent. However, cells exposed to a dual concentration gradient of both TNF-alpha and CSF-1 did migrate successfully, although the chemotactic response to CSF-1 was abolished.
Construction of mammalian genomic libraries using lambda replacement vectors.
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Osteopetrotic (op/op) mice deficient in macrophages have the ability to mount a normal T-cell-dependent immune response.
The osteopetrotic (op/op) mouse possesses an inactivating mutation in the CSF-1 gene that is associated with a lack of certain mononuclear phagocyte populations. To examine the effects of these deficiencies on T-cell-dependent immune functions, the responses of op/op and normal mice to a T-cell-dependent antigen, ovalbumin, and to a foreign histocompatibility antigen were compared. The ability of op/op mice (1) to induce antigen-specific proliferation of naive T-cells, (2) to generate cytotoxic T-lymphocytes, (3) to supply spleen cells to serve as stimulators in a mixed lymphocyte reaction, and (4) to produce IgM and IgG antibodies was indistinguishable from normal mice. These data are consistent with the involvement of specific macrophages, or other cells whose development is independent of CSF-1, in these immune responses.
Comparison of development of pig zygotes and embryos in simple and complex culture media.
The development of pig zygotes and embryos in three culture media was investigated. Zygotes and morulae, recovered from superovulated prepubertal gilts after slaughter, were randomly allocated to culture in one of three simple media: (i) Chatot, Ziomek, Bavister medium (CZB) in 5% CO2:95% air; (ii) Hepes-buffered CZB medium (H-CZB) in 100% air; or in a complex solution enriched with vitamins and amino acids; or (iii) modified Eagle's minimal essential medium (Pig-MEM) in 5% CO2:95% air. The relative effectiveness of the various media and gas combinations was assessed by determining the percentage of zygotes and morulae that developed into blastocysts and hatched from their zonae, as well as by the mean number of cells per embryo. Of zygotes cultured either in CZB in CO2:air, or in H-CZB in air alone, 65% and 57% developed into blastocysts, respectively; no zygotes cultured in Pig-MEM in CO2:air developed beyond the four-cell stage. After 115 h of culture, embryos resulting from zygotes cultured in either CZB or H-CZB contained approximately 50 cells per embryo. Morulae differed from zygotes in their developmental potential in the three media in that virtually all morulae cultured in CZB, H-CZB or Pig-MEM developed to the expanded blastocyst stage in vitro. However, of blastocysts resulting from morulae cultured in Pig-MEM, 97% hatched from their zonae, whereas only 8% and 5%, respectively, of those resulting from morulae cultured in CZB or H-CZB did so.(ABSTRACT TRUNCATED AT 250 WORDS)
Primary care: the driver in health care reform.
The changes occurring in the health care industry have resulted in a cost-quality competition that has not been present in the past. Because of this competition, managed care is a growing way of financing and providing health care to the people of the United States. Managed care depends heavily on competent primary care physicians. Because primary care physicians are in short supply, the status and financial rewards of primary care practice are increasing. The primary care physician will be the dominant force in medical practice in the immediate future. He or she is capable in a managed setting of resolving the perceived problems of the health care industry in responding to the drivers of health care reform. Costs are reduced while quality is maintained. Access to health care is improved, and fragmentation of health care is significantly lessened.
The role of colony-stimulating factor 1 and its receptor in the etiopathogenesis of endometrial adenocarcinoma.
Colony-stimulating factor 1 (CSF-1) is a homodimeric growth factor that humorally regulates the growth and differentiation of mononuclear phagocytes, and locally regulates maternal-fetal interactions during pregnancy. It exerts these actions through a transmembrane tyrosine kinase receptor, colony-stimulating factor 1 receptor (CSF-1R), the product of the c-fms proto-oncogene. Recent studies have demonstrated overexpression of CSF-1 and its receptor in breast, ovarian, and endometrial adenocarcinomas. To further investigate the possible role of CSF-1 and its receptor in the pathogenesis of endometrial adenocarcinoma, a prospective study was undertaken to study CSF-1 expression in benign and neoplastic endometrial epithelium and to compare serum CSF-1 levels in endometrial adenocarcinoma patients with healthy perimenopausal women. The mean serum levels of CSF-1 in 71 patients with endometrial cancer (4.9 +/- 1.8 microgram/liter) were significantly elevated compared with levels found in the 32 controls (3.5 +/- 1.1 microgram/liter). Within the endometrial adenocarcinoma group, circulating CSF-1 levels were significantly elevated in patients with large tumor volume, high grade, myometrial invasion, residual disease, and circulating CA-125 levels. High serum levels of serum CSF-1 were associated with elevated serum CA19-9 and CA-125 levels. Immunohistochemistry results revealed in tumor epithelium intense staining for CSF-1R (27 of 54 cases, 50%) and elevated staining for CSF-1 (41 of 54 cases, 75.9%), with intense staining of CSF-1 in 16 of 54 cases (29.6%). Staining was significantly greater in intensity and number of cells involved in malignant compared with benign epithelium for CSF-1R and CSF-1 (P = 0.05 and <0.0001, respectively). A positive correlation between amount and intensity of CSF-1 and CSF-1R staining in endometrial adenocarcinoma tissue was also demonstrated (P = 0.007). CSF-1 and CSF-1R mRNA was also detected in the tumor samples, confirming the expression of the protein in these tissues. Reverse transcription-PCR demonstrated the presence of mRNA for both the transmembrane and secreted forms of CSF-1 in all tumors analyzed. These results therefore support the hypotheses that CSF-1 and CSF-1R are overexpressed in endometrial adenocarcinoma, that levels of expression significantly correlate with clinicopathological risk factors for poor outcome, and that CSF-1 in association with its receptor via autocrine, juxtacrine, and/or paracrine interactions has a causal role in endometrial adenocarcinoma development and proliferation.
Colony stimulating factor 1 is required for mammary gland development during pregnancy.
The study of colony stimulating factor 1 (CSF-1), a homodimeric serum growth factor that regulates mononuclear phagocytes and is involved in maternal-fetal interactions during pregnancy, was dramatically enhanced by the observation that the recessive mutation osteopetrosis, op, is an inactivating mutation in the CSF-1-encoding gene. Homozygous mutant (op/op) mice completely lack CSF-1, are osteopetrotic consequent to a deficiency in osteoclasts, have severely reduced numbers of macrophages, and have reduced fertility evident at the pre- and postimplantation stages of pregnancy. We show here that op/op females have a lactational defect, and consequently, although some are able to produce offspring, few nurture any pups and none feeds a full litter. This lactational defect is due to incomplete mammary gland ductal growth during pregnancy, a precocious development of the lobulo-alveolar system, and despite expression of milk proteins, a failure to switch to a lactational state. These data show that CSF-1 has a role in the development of the mammary gland during pregnancy.
Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of the mouse.
Colony stimulating factor-1 (CSF-1) regulates the survival, proliferation and differentiation of mononuclear phagocytes. The osteopetrotic (op/op) mutant mouse is devoid of CSF-1 due to an inactivating mutation in the CSF-1 gene and is deficient in several mononuclear phagocyte subpopulations. To analyze more fully the requirement for CSF-1 in the establishment and maintenance of mononuclear phagocytes, the postnatal development of cells bearing the macrophage marker antigens F4/80 and MOMA-1, in op/op mice and their normal (+/op or +/+) littermates, were studied during the first three months of life. In normal mice, maximum expression of tissue F4/80+ cells was generally correlated with the period of maximum organogenesis and/or cell turnover. Depending on the tissue, the F4/80+ cell density either decreased, transiently increased or gradually increased with age. In op/op mice, tissues that normally contain F4/80+ cells could be classified into those in which F4/80+ cells were absent and those in which the F4/80+ cell densities were either reduced, normal or initially normal then subsequently reduced. To assess which F4/80+ populations were regulated by circulating CSF-1 in normal mice, op/op mice in which the circulating CSF-1 concentration was restored to above normal levels by daily subcutaneous injection of human recombinant CSF-1 from day 3 were analyzed. These studies suggest that circulating CSF-1 exclusively regulates both the F4/80+ cells in the liver, spleen and kidney and the MOMA-1+ metallophilic macrophages in the spleen. Macrophages of the dermis, bladder, bone marrow and salivary gland, together with a subpopulation in the gut, were partially restored by circulating CSF-1, whereas macrophages of the muscle, tendon, periosteum, synovial membrane, adrenals and the macrophages intimately associated with the epithelia of the digestive tract, were not corrected by restoration of circulating CSF-1, suggesting that they are exclusively locally regulated by this growth factor. Langerhans cells, bone marrow monocytes and macrophages of the thymus and lymph nodes were not significantly affected by circulating CSF-1 nor decreased in op/op mice, consistent with their regulation by other growth factors. These results indicate that important differences exist among mononuclear phagocytes in their dependency on CSF-1 and the way in which CSF-1 is presented to them. They also suggest that the prevalent role of CSF-1 is to influence organogenesis and tissue turnover by stimulating the production of tissue macrophages with local trophic and/or scavenger (physiological) functions. Macrophages involved in inflammatory and immune (pathological) responses appear to be dependent on other factors for their ontogenesis and function.(ABSTRACT TRUNCATED AT 400 WORDS)
The in vivo isotopic labeling of proteins for polyacrylamide gel electrophoresis.
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Two-dimensional polyacrylamide gel electrophoresis of proteins.
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Efficient DNA transfection of quiescent mammalian cells using poly-L-ornithine.
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