Biomedical subjects

J W Pollard

Publications and source records attributed to J W Pollard.

At least 19 recordsLinked to original sources

PubMed2002

Maturation status, protein synthesis and developmental competence of oocytes derived from lambs and ewes.

The efficacy of oocyte selection for in vitro embryo production depends on the abundance and diameter of follicles, cumulus layers around the oocytes and subsequent fertilization. Application of 'ovum pick-up' technique allows us to utilize partially matured oocytes for embryo production even from juvenile subjects. To compare their developmental competence, oocytes derived from lambs and ewes and cultured in maturation medium for up to 26 h were assessed at 2 h intervals by confocal microscopy after chromatin and microtubulin-specific fluorochrome labelling. Lamb oocytes reached second meiotic metaphase (MII) at lower numbers at 24 h (60.0%) and 26 h (28.6%) whereas 85.7% of adult-derived oocytes attained MII status by 24 h of maturation. Radiolabelling of oocyte proteins revealed higher incorporation of [(35)S-]-methionine and [(35)S]-cysteine in adult-derived oocytes compared to lamb oocytes. Although the cleavage rate of lamb oocytes was similar to that of ewe oocytes, the proportion reaching blastocyst stage was significantly lower (p < 0.05) in the lamb-derived oocytes. However, blastocysts from both types of oocytes displayed similar cell lineage allocations to inner cell mass and trophectoderm.

Animals↗

PubMed2001

Rescue of the colony-stimulating factor 1 (CSF-1)-nullizygous mouse (Csf1(op)/Csf1(op)) phenotype with a CSF-1 transgene and identification of sites of local CSF-1 synthesis.

Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. It is expressed as a secreted glycoprotein or proteoglycan found in the circulation or as a biologically active cell-surface glycoprotein. To investigate tissue CSF-1 regulation, CSF-1-null Csf1(op)/Csf1(op) mice expressing transgenes encoding the full-length membrane-spanning CSF-1 precursor driven by 3.13 kilobases of the mouse CSF-1 promoter and first intron were characterized. Transgene expression corrected the gross osteopetrotic, neurologic, weight, tooth, and reproductive defects of Csf1(op)/Csf1(op) mice. Detailed analysis of one transgenic line revealed that circulating CSF-1, tissue macrophage numbers, hematopoietic tissue cellularity, and hematopoietic parameters were normalized. Tissue CSF-1 levels were normal except for elevations in 4 secretory tissues. Skin fibroblasts from the transgenic mice secreted normal amounts of CSF-1 but also expressed some cell-surface CSF-1. Also, lacZ driven by the same promoter/first intron revealed beta-galactosidase expression in hematopoietic, reproductive, and other tissue locations proximal to CSF-1 cellular targets, consistent with local regulation by CSF-1 at these sites. These studies indicate that the 3.13-kilobase promoter/first intron confers essentially normal CSF-1 expression. They also pinpoint new cellular sites of CSF-1 expression, including ovarian granulosa cells, mammary ductal epithelium, testicular Leydig cells, serous acinar cells of salivary gland, Paneth cells of the small intestine, as well as local sites in several other tissues.

Animals↗

PubMed2001

Tumour-stromal interactions. Transforming growth factor-beta isoforms and hepatocyte growth factor/scatter factor in mammary gland ductal morphogenesis.

The mammary gland undergoes morphogenesis through the entire reproductive life of mammals. In mice, ductal outgrowth from the nipple across the fat pad results in an intricate, well spaced ductal tree that further ramifies and develops alveolar structures during pregnancy. Ductal morphogenesis is regulated by the concerted action of circulating steroid and polypeptide hormones, and local epithelial-mesenchymal inductive signals. Transforming growth factor (TGF)-beta1-3 and hepatocyte growth factor (HGF)/scatter factor (SF) are important components of this latter signaling pathway. TGF-beta1 and TGF-beta3 have roles in both promotion and inhibition of branching morphogenesis that are dependent on concentration and context. HGF/SF promotes ductal outgrowth and tubule formation in the mammary gland. These data suggest that these two growth factors have complementary roles in promoting mammary ductal morphogenesis and in maintaining ductal spacing. In addition, TGF-beta3 triggers apoptosis in the alveolar epithelia, which is a necessary component of mammary gland involution and return of the ductal structure to a virgin-like state after lactation.

Animals↗

PubMed2001

Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy.

In human breast carcinomas, overexpression of the macrophage colony-stimulating factor (CSF-1) and its receptor (CSF-1R) correlates with poor prognosis. To establish if there is a causal relationship between CSF-1 and breast cancer progression, we crossed a transgenic mouse susceptible to mammary cancer with mice containing a recessive null mutation in the CSF-1 gene (Csf1(op)) and followed tumor progression in wild-type and null mutant mice. The absence of CSF-1 affects neither the incidence nor the growth of the primary tumors but delayed their development to invasive, metastatic carcinomas. Transgenic expression of CSF-1 in the mammary epithelium of both Csf1(op)/Csf1(op) and wild-type tumor-prone mice led to an acceleration to the late stages of carcinoma and to a significant increase in pulmonary metastasis. This was associated with an enhanced infiltration of macrophages into the primary tumor. These studies demonstrate that the growth of mammary tumors and the development to malignancy are separate processes and that CSF-1 selectively promotes the latter process. CSF-1 may promote metastatic potential by regulating the infiltration and function of tumor-associated macrophages as, at the tumor site, CSF-1R expression was restricted to macrophages. Our data suggest that agents directed at CSF-1/CSF-1R activity could have important therapeutic effects.

Animals↗

PubMed2001

Regulation of meiotic recombination and prophase I progression in mammals.

Meiosis is the process by which diploid germ cells divide to produce haploid gametes for sexual reproduction. The process is highly conserved in eukaryotes, however the recent availability of mouse models for meiotic recombination has revealed surprising regulatory differences between simple unicellular organisms and those with increasingly complex genomes. Moreover, in these higher eukaryotes, the intervention of physiological and sex-specific factors may also influence how meiotic recombination and progression are monitored and regulated. This review will focus on the recent studies involving mouse mutants for meiosis, and will highlight important differences between traditional model systems for meiosis (such as yeast) and those involving more complex cellular, physiological and genetic criteria.

Animals↗

PubMed2001

Pigs expressing salivary phytase produce low-phosphorus manure.

To address the problem of manure-based environmental pollution in the pork industry, we have developed the phytase transgenic pig. The saliva of these pigs contains the enzyme phytase, which allows the pigs to digest the phosphorus in phytate, the most abundant source of phosphorus in the pig diet. Without this enzyme, phytate phosphorus passes undigested into manure to become the single most important manure pollutant of pork production. We show here that salivary phytase provides essentially complete digestion of dietary phytate phosphorus, relieves the requirement for inorganic phosphate supplements, and reduces fecal phosphorus output by up to 75%. These pigs offer a unique biological approach to the management of phosphorus nutrition and environmental pollution in the pork industry.

6-Phytase↗

PubMed2001

Aberrant macrophage and neutrophil population dynamics and impaired Th1 response to Listeria monocytogenes in colony-stimulating factor 1-deficient mice.

Listeria monocytogenes, a facultative intracellular bacterium, has been used extensively to study innate immune responses. Macrophages act as hosts for this bacterium as well as a major defense against it. Using mice homozygous for a null mutation (Csf1(op)) in the gene for the mononuclear phagocytic growth factor colony-stimulating factor 1 (CSF-1), we have demonstrated that CSF-1-regulated macrophages were essential to defend against a listerial infection. In the absence of CSF-1, monocytes were not recruited to the sites of infection due to the lack of synthesis of the macrophage chemoattractant chemokine MCP-1. In addition, there was no burst of interleukin-10 (IL-10) synthesis that has been shown to result in the egress of neutrophils from sites of infection. Consequently, neutrophils were not replaced by macrophages, and numerous neutrophil-filled microabscesses developed, followed by tissue destruction and death of the mice. In the CSF-1 nullizygous mice compared to wild-type mice, there was also a very low synthesis of gamma interferon (IFN-gamma), resulting in reduced macrophage activation. However, the concentrations of the IFN-gamma-inducing cytokines IL-12 and IL-18 at this bacterial load were similar in these mutant mice. In contrast, IL-6 concentrations were dramatically reduced. Administration of IL-6 to Csf1(op)/Csf1(op) mice significantly increased the synthesis of IFN-gamma and reduced the bacterial burden to a greater extent than treatment with IFN-gamma alone. These data indicate that IL-6 occupies a central role in the CSF-1-regulated macrophage response to L. monocytogenes.

Abscess↗

PubMed2001

Genetic evidence for the interactions of cyclin D1 and p27(Kip1) in mice.

The cell cycle of cultured cells appears to be regulated by opposing actions of the cyclins together with their partners, the cyclin-dependent kinases (Cdk), and their inhibitors (Cki). Consistent with this situation null mutations in the genes for cyclin D1 and Cki p27(Kip1) in mice give opposite phenotypes of dwarfism and gigantism. To test their genetic interactions, we generated mice nullizygous for both genes. Correction of cyclin D1 or p27 null to wild-type phenotypes was observed for many but not all traits. These included, for cyclin D1(-/-) mice, body weight, early lethality, retinal hypoplasia, and male aggressiveness and, for p27(-/-) mice, body weight, retinal hyperplasia, and embryo implantation. p27(-/-) traits that were not corrected were the aberrant estrus cycles, luteal cell proliferation, and susceptibility to pituitary tumors. This mutual correction of these phenotypes is the first genetic demonstration of the interaction of these inhibitory and stimulatory cell cycle-regulatory molecules in vivo. The molecular basis for the correction was analyzed in the neonatal retina. Retinal cellularity was rescued in the cyclin D1 null mouse by loss of p27 with only a partial restoration of phosphorylation of retinoblastoma protein (Rb) and Cdk4 activity but with a dramatic elevation of Cdk2 activity. Our data provide in vivo genetic validation of cell culture experiments that indicated that p27 acts as a negative regulator of cyclin E-Cdk2 activity and that it can be titrated away by cyclin D-Cdk4 complexes. It also supports the suggestion that the cyclin E/Cdk2 pathway can largely bypass Rb in regulating the cell cycle in vivo.

Animals↗

PubMed2001

Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18(Ink4c) and p19(Ink4d).

Male mice lacking both the Ink4c and Ink4d genes, which encode two inhibitors of D-type cyclin-dependent kinases (Cdks), are infertile, whereas female fecundity is unaffected. Both p18(Ink4c) and p19(Ink4d) are expressed in the seminiferous tubules of postnatal wild-type mice, being largely confined to postmitotic spermatocytes undergoing meiosis. Their combined loss is associated with the delayed exit of spermatogonia from the mitotic cell cycle, leading to the retarded appearance of meiotic cells that do not properly differentiate and instead undergo apoptosis at an increased frequency. As a result, mice lacking both Ink4c and Ink4d produce few mature sperm, and the residual spermatozoa have reduced motility and decreased viability. Whether or not Ink4d is present, animals lacking Ink4c develop hyperplasia of interstitial testicular Leydig cells, which produce reduced levels of testosterone. The anterior pituitary of fertile mice lacking Ink4c or infertile mice doubly deficient for Ink4c and Ink4d produces normal levels of luteinizing hormone (LH). Therefore, the failure of Leydig cells to produce testosterone is not secondary to defects in LH production, and reduced testosterone levels do not account for infertility in the doubly deficient strain. By contrast, Ink4d-null or double-null mice produce elevated levels of follicle-stimulating hormone (FSH). Because Ink4d-null mice are fertile, increased FSH production by the anterior pituitary is also unlikely to contribute to the sterility observed in Ink4c/Ink4d double-null males. Our data indicate that p18(Ink4c) and p19(Ink4d) are essential for male fertility. These two Cdk inhibitors collaborate in regulating spermatogenesis, helping to ensure mitotic exit and the normal meiotic maturation of spermatocytes.

Animals↗

PubMed2001

Eotaxin is required for eosinophil homing into the stroma of the pubertal and cycling uterus.

The presence of eosinophils in the endometrium of rodents during the estrous cycle or after E2 administration to ovariectomized animals is well documented. Nevertheless, the chemoattractant for eosinophils and the function of E-dependent eosinophils during the estrous cycle remain unknown. Using mice homozygous for a null mutation in the gene for eotaxin, a specific chemokine for eosinophils, we have identified eotaxin as being necessary for eosinophil homing into the uterine stroma, and regulated by E2 during the estrous cycle. In the absence of eosinophils, the onset of estrous cycle displayed a 2-wk delay along with the first age of parturition, suggesting a possible local role of eosinophils present in the pubertal uterus in preparing the mature uterus for pregnancy. However, despite the absence of eosinophils, once the mice reach maturity, their estrous cycles as well as their reproductive functions were normal. Our results demonstrate that E2 acts through eotaxin to recruit eosinophils to the uterine stroma during the estrous cycle in mice, but that these cells do not have a function in regulating either the duration of the estrous cycle or fertility of mice.

Animals↗

PubMed2001

Intranuclear endoplasmic reticulum induced by Nopp140 mimics the nucleolar channel system of human endometrium.

Exogenous expression of the characteristic repeat domain of the nucleolar chaperone Nopp140 induces the formation of intranuclear structures, termed R-rings. Here, the R-rings are identified as extensive stacks of membrane cisternae in the otherwise membrane-free nucleus. They consist of bona fide endoplasmic reticulum (ER) containing integral membrane proteins of the smooth and rough ER. Although lacking nuclear pore complexes and lamina, the R-rings derive specifically from the inner nuclear membrane. These findings are consistent with the idea that all transmembrane proteins synthesized in the ER and the outer nuclear membrane can freely diffuse through the pore membrane domain into the inner membrane. Uniquely, the soluble transfected Nopp140 is directly involved in the generation of these membrane stacks as it localizes to the electron dense matrix in which they are embedded. The only well-documented example of intranuclear membrane proliferation is the nucleolar channel system of the postovulation human endometrium. The transient emergence of the nucleolar channel system correlates precisely with the readiness of the endometrium for the implantation of the fertilized egg. The nucleolar channel system exhibits an ultrastructure that is indistinguishable from R-rings, and nuclei of human endometrium harbor Nopp140 and ER marker containing structures. Therefore, the nucleolar channel system appears to be identical to the R-rings, suggesting a role for Nopp140 in human reproduction.

Animals↗

PubMed2000

MutS homolog 4 localization to meiotic chromosomes is required for chromosome pairing during meiosis in male and female mice.

Msh4 (MutS homolog 4) is a member of the mammalian mismatch repair gene family whose members are involved in postreplicative DNA mismatch repair as well as in the control of meiotic recombination. In this report we show that MSH4 has an essential role in the control of male and female meiosis. We demonstrate that MSH4 is present in the nuclei of spermatocytes early in prophase I and that it forms discrete foci along meiotic chromosomes during the zygotene and pachytene stages of meiosis. Disruption of the Msh4 gene in mice results in male and female sterility due to meiotic failure. Although meiosis is initiated in Msh4 mutant male and female mice, as indicated by the chromosomal localization of RAD51 and COR1 during leptonema/zygonema, the chromosomes fail to undergo normal pairing. Our results show that MSH4 localization on chromosomes during the early stages of meiosis is essential for normal chromosome synapsis in prophase I and that it acts in the same pathway as MSH5.

Animals↗

PubMed2000

The trophoblast is a component of the innate immune system during pregnancy.

Systemic infection with Listeria monocytogenes, a Gram-positive intracellular bacterium, has been used extensively to analyze the innate immune response. Macrophages are central to this response, acting as both the host for and principal defense against this bacterium. During pregnancy L. monocytogenes has a predilection for replication at the maternal-placental interface and consequently is an important cause of fetal morbidity and mortality. However, macrophages are mostly excluded from the murine placenta with neutrophils acting as the main immune effector cell against this bacterium. Colony stimulating factor (CSF)-1, a macrophage growth factor, is synthesized in high concentrations by the uterine epithelium during pregnancy, where it is targeted to trophoblast bearing CSF-1-receptors. To define the involvement of CSF-1 in placental immunity, we infected pregnant mice either homozygous or heterozygous for an inactivating recessive mutation in the gene for CSF-1 (osteopetrotic; Csfmop) with L. monocytogenes. CSF-1 was required to recruit neutrophils to the site of listerial infection in the decidua basalis, and infection by Listeria remained unrestrained in its absence. CSF-1 acted by inducing the trophoblast to synthesize the neutrophil chemoattractants (KC) and macrophage inflammatory protein (MIP)-2. Thus, during pregnancy, trophoblast responsive to CSF-1 acts to organize the maternal immune response to bacterial infection at the utero-placental interface. This previously unknown function indicates that the trophoblast acts as a pregnancy-specific component of the innate immune system.

Animals↗

PubMed2000

Postnatal mammary gland development requires macrophages and eosinophils.

Interactions between mammary epithelial and mesenchymal cells including fibroblasts and adipocytes are crucial for the proper postnatal development of the mammary ductal tree. Often overlooked, however, are the migrant cells that enter tissues at different stages of development. In this paper we identify two such cell types, macrophages and eosinophils, that are recruited around the growing terminal end buds (TEBs) during postnatal development. An important role for leukocytes in mammary gland ductal outgrowth is first demonstrated by depleting mice of leukocytes using sub-lethal (gamma)-irradiation. This treatment results in a curtailment of mammary gland epithelial development that is completely rescued by bone-marrow transplantation, concurrent with a restoration of macrophage and eosinophil recruitment around the growing ducts. Using mice homozygous for a null mutation in the gene for CSF1 (Csfm(op)/Csfm(op)), the major growth factor for macrophages, we show that in the absence of CSF1, the population of macrophages in mammary glands is depleted. In this mutant, the formation of TEBs, their outgrowth into the fat pad and the branching of the resultant ducts are all impaired. Similarly, by using mice homozygous for a null mutation in the gene for eotaxin, a major chemokine for local recruitment of eosinophils in tissue, we identify eotaxin as the necessary and sufficient chemokine responsible for eosinophil recruitment around TEBs. In the absence of eosinophils, mammary gland branch formation and to a lesser extent TEB formation are reduced. Our data show that CSF1-regulated macrophages, in collaboration with eotaxin-regulated eosinophils, have essential and complementary functions in regulating the branching morphogenesis of the mammary gland.

Animals↗

PubMed2000

Transforming growth factor beta3 induces cell death during the first stage of mammary gland involution.

Involution of the mammary gland following weaning is divided into two distinct phases. Initially, milk stasis results in the induction of local factors that cause apoptosis in the alveolar epithelium. Secondly after a prolonged absence of suckling, the consequent decline in circulating lactogenic hormone concentrations initiates remodeling of the mammary gland to the virgin-like state. We have shown that immediately following weaning TGFbeta3 mRNA and protein is rapidly induced in the mammary epithelium and that this precedes the onset of apoptosis. Unilateral inhibition of suckling and hormonal reconstitution experiments showed that TGFbeta3 induction is regulated by milk stasis and not by the circulating hormonal concentration. Directed expression of TGFbeta3 in the alveolar epithelium of lactating mice using a beta-lactoglobulin promoter mobilized SMAD4 translocation to the nucleus and caused apoptosis of these cells, but not tissue remodeling. Transplantation of neonatal mammary tissue derived from TGFbeta3 null mutant mice into syngenic hosts resulted in a significant inhibition of cell death compared to wild-type mice upon milk stasis. These results provide direct evidence that TGFbeta3 is a local mammary factor induced by milk stasis that causes apoptosis in the mammary gland epithelium during involution.

Animals↗

PubMed2000

Production of sexed lambs after biopsy of ovine blastocyts produced in vitro.

Embryos were generated by in vitro fertilization of in vitro-matured oocytes, cultured to the blastocyst stage, biopsied for sex determination by a PCR-based procedure, and transferred to synchronized recipients. Three out of 5 sheep (60%) were diagnosed pregnant, and 4 lambs of predicted sex were born.

Animals↗

PubMed1999

Macrophages: important accessory cells for reproductive function.

Macrophages are found throughout reproductive tissues. To determine their role(s), we have studied mice homozygous for a null mutation (Csfm(op)) in the gene encoding the major macrophage growth factor, colony-stimulating factor-1 (CSF-1). Both male and female Csfm(op)/Csfm(op) mice have fertility defects. Males have low sperm number and libido as a consequence of dramatically reduced circulating testosterone. Females have extended estrous cycles and poor ovulation rates. CSF-1 is the principal growth factor regulating macrophage populations in the testis, male accessory glands, ovary, and uterus. However, analyses of CSF-1 nullizygous mice suggest that the primary reproductive defect is in the development of feedback regulation of the hypothalamic-pituitary axis. Although not correlating with deficiencies of microglia populations, electrophysiological investigations indicate an impairment of neuronal responses. This suggests that microglia, under the influence of CSF-1, act to organize neuronal connectivity during development and that the absence of this function results in a perturbation of the hypothalamic-pituitary-gonadal axis. Macrophages also appear to have functions in the differentiated tissues of the reproductive system, including having a positive influence on steroidogenic cells. These data suggest that macrophages, through their trophic functions, can be considered as essential accessory cells for normal reproductive functioning.

Animals↗

PubMed1999

Colony-stimulating factor-1 and its receptor do not have a role in the pathogenesis of uterine sarcomas.

OBJECTIVE: Several studies have demonstrated overexpression of the mononuclear phagocytic growth factor colony-stimulating factor-1 (CSF-1) and its receptor (CSF-1R) in breast, ovarian, and endometrial adenocarcinomas, and their expression in each of these cancers is strongly correlated with poor prognosis. In addition to adenocarcinomas, sarcomas that are highly malignant arise at much lower frequency in the uterus. Given the common organ of origin and hormonal environment of the adenocarcinomas, we evaluated the potential role of CSF-1 and CSF-1R in the genesis of these tumors using immunohistochemical methods. RESULTS: Immunohistochemical analysis was performed on 19 archival uterine sarcoma samples. Affinity-purified rabbit anti-CSF-1 antiserum (R52) and human cross-reactive murine anti-c-fms antibody were used. In the 19 cases evaluated for CSF-1 immunoreactivity, 42.1% had staining in less than 25% of the tumor, 36.9% had staining in 25-50% of the tumor, and only 21% had staining in greater than 50% of the tumor. When present, the majority of the CSF-1 immunostaining was associated with the extracellular matrix. There was variable intensity in CSF-1 expression: 52.6% had negative to mild staining, and 47.4% had moderate to strong staining. Immunostaining for the CSF-1R revealed that 52.6% of tumors had expression in less than 25% of cells, 21.0% had expression in 25-50% of the tumor, and 26.4% had staining in greater than 50% of the tumor. There was variable intensity of CSF-1R staining. Slight staining was found in 31.6% of the cases, moderate staining was found in 47.4% of the tumors, and 21.0% of the cases had strong expression. There was no statistically significant correlation between CSF-1 and CSF-1R expression and stage, estrogen/progesterone receptor status, number of mitoses per 10 high-power fields, or disease outcome. In addition, overall expression and intensity of CSF-1 and CSF-1R did not predict tumor virulence or disease outcome. CONCLUSION: In contradistinction to endometrial adenocarcinomas, in which CSF-1/CSF-1R is strongly correlated with tumor progression, CSF-1 and CSF-1R overexpression does not appear to play a role in the growth and differentiation of uterine sarcomas.

Female↗