F Pothier

Laval University, Quebec City, Quebec, Canada

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Publications (20)132.97 Total impact

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    ABSTRACT: It is generally accepted that mammalian females are born with a finite pool of oocytes and that this is the sole source of ovules throughout the reproductive life of the adult. This dogma was shaken in 2003 when researchers showed that the oocyte stock might be renewable in adult mammals. It has been proposed that hematopoietic stem cells might be a source of new oocytes. These discoveries have puzzled many researchers and remain controversial. In our study, we attempted to determine if transplanted bone marrow cells could provide new oocytes in PU.1 mice and in severe combined immunodeficiency (SCID) mice after treatment with chemotherapeutic agents. We also examined the possibility that grafted bovine embryonic ovarian cortex might provide an environment favoring such a response. We found no evidence that transplanted bone marrow cells provide new fertilizable oocytes in PU.1 mice, in SCID mice treated with chemotherapeutic agents, or with bovine embryonic ovarian tissue grafts. However, transplanted bone marrow cells have improved the fertility of SCID mice previously treated with chemotherapeutic agents. These data suggest that bone marrow cells cannot provide new oocytes but can positively influence ovarian physiology to improve the fertility of mice previously treated with chemotherapeutic agents.
    04/2012; 14(2):123-9. DOI:10.1089/cell.2011.0066
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    ABSTRACT: Previous studies have shown that using nonspecific phosphodiesterase (PDE) inhibitors such as caffeine improved milk production, supporting the premise that modulation of intracellular concentration of cyclic nucleotides (cyclic AMP, cyclic guanosine 3'-5'-monophosphate) is involved. Intracellular cyclic nucleotides are degraded by the PDE enzyme family. The contribution of type IV PDE (PDE4) in the secretion of casein has been reported in rat mammary gland. The objective of this study was to demonstrate the functional presence of the PDE4 family in the bovine mammary gland. To understand the enzymatic expression pattern in the mammary gland, tissue samples were taken randomly from udders obtained from a local slaughterhouse. Reverse transcription PCR revealed that the PDE4D transcript was amplified, and the expected size fragment was obtained in a 1% agarose gel. Sequence analysis of the amplicon resulted in 99% homology to PDE4D. Moreover, Western blotting using a specific PDE4D antibody has confirmed that the protein of the isoenzyme PDE4D1 is present. A clear immunoreactive signal was also observed within the acini where epithelial cells are located. Assaying cyclic AMP PDE activity reported a total activity of 38.71 +/- 3.22 fmol/min per microg of total protein. Rolipram, a specific PDE4 inhibitor, showed a sensitive activity of 8.48 +/- 1.28 fmol/min per microg of total protein, indicating that PDE4 is responsible for one-fifth of the total enzymatic activity of PDE in the mammary gland. To further validate the presence of PDE4D in the bovine mammary epithelial cells, protein extracts from bovine mammary epithelial cells were separated on SDS-PAGE gels, and PDE4D protein was detected. The PDE assays reported a total activity of 30.16 +/- 4.82 fmol/min per microg of total protein. Rolipram showed a sensible activity of 11.91 +/- 5.93 fmol/min per microg of total protein. In conclusion, these results not only demonstrate the presence of PDE4D transcript and protein, but also show an active enzyme, suggesting a functional role of PDE4D in bovine mammary gland.
    Journal of Dairy Science 09/2009; 92(8):3757-65. DOI:10.3168/jds.2009-2065 · 2.57 Impact Factor
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    ABSTRACT: Cloning of mammalian oocytes requires that the recipient oocyte is enucleated to remove all genetic material associated with the chromosomes. The procedure currently used in most species requires careful micromanipulation of oocytes treated with cytochalasin B to prevent structural damage. Although functional, this procedure requires time and limits the number of oocytes available for cloning, and our ability to understand the mechanisms of nuclear reprogramming. Therefore, this study aimed at evaluating different procedures to enucleate large pools of oocytes in a time-efficient manner. Two different approaches were tested. The first approach involved centrifugation of zona-free oocytes through a percoll gradient to separate the portion containing the chromatin from the cytoplasmic portion. The second used etoposide to prevent chromatin segregation at first metaphase and resulting in the expulsion of all chromosomes in the polar body. Using the chemical approach an average enucleation rate of 39.4 +/- 7.5% was obtained, while the centrifugation approach resulted in an average enucleation rate of 66.9 +/- 6. In terms of time efficiency, the control manipulation method takes 0.11 min and the centrifugation took an average of 0.52 min per oocyte. The MPF activity at the end of procedure was estimated through the measurement of H1 activity and as expected, the etoposide-cycloheximide treated oocytes had lower H1 activity which was restored by further incubation in the maturation medium for 5 hr while the centrifugation gave a nonsignificant intermediary result. In conclusion, the results presented suggest that both the chemical and the mechanical methods are usable alternatives to micromanipulation of oocytes to generate a large number of chromosome free cytoplasm for biochemical analysis. Mol. Reprod. Dev. 67: 70-76, 2004.
    Molecular Reproduction and Development 02/2004; 67(1):70-6. DOI:10.1002/mrd.20011 · 2.53 Impact Factor
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    Michael K Dyck · Dan Lacroix · François Pothier · Marc-André Sirard
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    ABSTRACT: Transgenic animal bioreactors represent a powerful tool to address the growing need for therapeutic recombinant proteins. The ability of transgenic animals to produce complex, biologically active recombinant proteins in an efficient and economic manner has stimulated a great deal of interest in this area. As a result, genetically modified animals of several species, expressing foreign proteins in various tissues, are currently being developed. However, the generation of transgenic animals is a cumbersome process and remains problematic in the application of this technology. The advantages and disadvantages of different transgenic systems in relation to other bioreactor systems are discussed.
    Trends in Biotechnology 10/2003; 21(9):394-9. DOI:10.1016/S0167-7799(03)00190-2 · 11.96 Impact Factor
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    ABSTRACT: Insulin-like growth factor-I (IGF-I) has been implicated in a wide variety of physiological processes including ovarian function. To better understand the ovarian role of IGF-I, transgenic mice harbouring a human IGF-I cDNA (hIGF-I) under the control of the mouse LH receptor promoter were generated. Expression of the hIGF-I, determined by Northern blot, was found to occur in the gonad tissues of these transgenic mice. The hIGF-I protein was also detectable by radioimmunoassay in ovarian extracts as well as in the plasma. The fertility of mating transgenic females, as estimated by the number of implantation sites post-coitum, did not appear to be affected. However, transgenic females who failed to mate and produce offspring were found to possess polycystic ovaries. Evaluation of testosterone, estradiol, and LH levels revealed that transgenic animals had significantly elevated circulating levels of testosterone compared to their non-transgenic littermates, while LH levels in transgenic females were significantly lower. Yet, estradiol appeared to be unaffected. These results support the contention that the IGF system plays an important role in ovarian function and that an imbalance in this system may result in ovarian pathology.
    Molecular Reproduction and Development 07/2001; 59(2):178-85. DOI:10.1002/mrd.1020 · 2.53 Impact Factor
  • Anne Rieth · Francois Pothier · Marc‐André Sirard
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    ABSTRACT: There are several methods of modifying bovine genomes. Pronuclear microinjection is more widely used but it is still to be improved. Searches for alternatives have lead to the development of new methods including SMGT (Sperm Mediated Gene Transfer), in which live spermatozoa are used as vehicles for DNA delivery during in vitro fertilization. In previous studies, we presented evidence that a highly repetitive Alu-like repeat favours transgenesis by homologous recombination (HR). Up to 60% integration via HR was obtained following pronuclear microinjection of a Pst1 beta-actin GFP DNA construction. In the present study, we show that HR-mediated integration is also possible using SMGT, since bovine spermatozoa electroporated with the same DNA construct are able to transfer it to a high proportion of embryos obtained by in vitro fertilization. Swim-up selected bovine spermatozoa were mixed with the Pst1 beta-actin GFP construct (6 x 10(6) spermatozoids were incubated with 600 ng of muDNA), submitted or not to electroporation (300 V, 25 F) and treated or not with DNase I. The process of electroporation itself did not affect in vitro embryonic development. However, oocytes fertilized with electroporated DNA-treated spermatozoa developed beyond the 16-cell stage in proportions that were significantly lower (27% with Pst1 beta-actin GFP and 34% with beta-actin GFP) compared to the control without DNA (44%). On the other side, the use of electroporation significantly increased the uptake of DNA. The number of homologous recombination events detected by PCR went from 3.5% without electroporation to 46.5% after electroporation. In conclusion, our results confirm that spermatozoa electroporation combined with homologous recombination in a highly repetitive Pst1 sequence is a feasible method to obtain transgenic bovine embryos.
    Molecular Reproduction and Development 01/2001; 57(4):338-45. DOI:10.1002/1098-2795(200012)57:4<338::AID-MRD5>3.0.CO;2-K · 2.53 Impact Factor
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    ABSTRACT: Production of foreign proteins in the tissues of transgenic animals represents an efficient and economical method of producing therapeutic and pharmaceutical proteins. In this study, we demonstrate that the mouse P12 gene promoter specific to the male accessory sex gland can be used to generate transgenic mice that express human growth hormone (hGH) in their seminal vesicle epithelium. The hGH is secreted into the ejaculated seminal fluids with the seminal vesicle lumen contents containing concentrations of up to 0.5 mg/ml. As semen is a body fluid that can be collected easily on a continuous basis, the production of transgenic animals expressing pharmaceutical proteins into their seminal fluid could prove to be a viable alternative to use of the mammary gland as a bioreactor.
    Nature Biotechnology 12/1999; 17(11):1087-90. DOI:10.1038/15067 · 41.51 Impact Factor
  • Anne Rieth · Francois Pothier · Marc Gagné · Marc‐ André Sirard
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    ABSTRACT: Homologous recombination (HR) has proven to be functional in mammalian embryos. The efficiency of the HR process was tested in bovine zygotes in an attempt to increase the frequency of transgene integration using different lengths of a bovine satellite (BS) DNA flanking both ends of a neo gene marker (called BS500, BS250, and BS50) and neo alone as a control. Pronuclear microinjection at 16-19 hr post insemination (hpi) of the BS500, BS250, BS50 or neo fragments at a concentration of 1 ng/microl resulted in an increasingly negative effect on embryo development. Therefore all microinjections were performed at a single molecular concentration (320 x 10(6) molecules/ microl). After microinjection, the embryos were allowed to develop for 6 days followed by morphological and PCR analysis. The HR event was detected by PCR in 13 of the 26 embryos (43%) that developed beyond the 12-cell stage, 7/22 (31%), 9/27 (33%), and 0/25 (0%) with the BS500, BS250, BS50, and neo constructs respectively. The length of BS homology had no effect on transgene integration. However, embryos injected with BS neo constructs had significantly lower development rates than neo injected zygotes (17% more than 16 cells for BS500; 14% for BS250; 16% for BS50 compared to 32% for neo, P < 0.05, 6 replicates). These results demonstrate that BS sequences have a negative effect on embryo development and survival regardless of the amount of DNA injected. The use of HR with highly repetitive genomic sequences is therefore a feasible procedure to produce transgenic bovine embryos.
    Molecular Reproduction and Development 05/1999; 53(1):1-7. DOI:10.1002/(SICI)1098-2795(199905)53:1<1::AID-MRD1>3.0.CO;2-7 · 2.53 Impact Factor
  • E Bélanger · N Jean · M Ouellet · R Sullivan · F Pothier
    Theriogenology 01/1999; 51(1):416-416. DOI:10.1016/S0093-691X(99)91975-1 · 1.80 Impact Factor
  • M. K Dyck · M Ouellet · M Gagné · F Pothier
    Theriogenology 01/1999; 51(1):419-419. DOI:10.1016/S0093-691X(99)91978-7 · 1.80 Impact Factor
  • Theriogenology 01/1999; 51(1):424-424. DOI:10.1016/S0093-691X(99)91983-0 · 1.80 Impact Factor
  • A Rieth · M Gagné · F Pothier
    Theriogenology 01/1999; 51(1):425-425. DOI:10.1016/S0093-691X(99)91984-2 · 1.80 Impact Factor
  • Marc Gagné · François Pothier · Marc-André Sirard
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    ABSTRACT: Microinjection into bovine zygotes was performed to evaluate the effects of the timing of injection during the phase of DNA replication on the subsequent in vitro development of embryos and expression of injected chicken β-actin promoter-lac Z gene construct. The period of DNA replication of bovine zygotes, determined by 3H-thymidine incorporation, begins between 12 hr and 13 hr postinsemination (hpi) of in vitro matured oocytes, reaches a maximum from 17 hpi to 19 hpi, and is complete by 21–22 hpi. Aphidicolin, an inhibitor of DNA polymerase alpha, was used to synchronize the pronuclei and the zygote population. Treatment with aphidicolin at 9–18 hpi arrested DNA replication without affecting formation of the pronuclei or embryo development. Cycloheximide, an inhibitor of protein synthesis, was used for nucleocytoplasmic resynchronization of the aphidicolin-treated zygotes. Microinjection was performed at 15 (early), 18 (mid), and 21 (late S phase) hpi. Embryonic development was affected following each of the three microinjection times. The development of zygotes injected at 18 hpi was significantly higher (P<0.01) after 5 days of culture than those injected at 15 hpi or 21 hpi. Expression of the marker gene was observed in the higher stage of development (>16 cells) only in the zygotes injected at 18 hpi. At the earlier stages of development, the proportions of embryos showing expression of the foreign gene were the same for all microinjection times. In aphidicolin- and cycloheximide-treated zygotes, expression of the marker gene followed the same curve as development, i.e., expression was low when injected early or late and higher (P<0.005) when injected in the middle of zygotic S phase. The ability of the embryos to survive microinjection and to express the marker gene as a function of hpi seems to be influenced mostly in the cytoplasm processing stage rather than the pronuclei processing stage. © 1995 Wiley-Liss, Inc.
    Molecular Reproduction and Development 05/1995; 41(2):184 - 194. DOI:10.1002/mrd.1080410209 · 2.53 Impact Factor
  • M Gagné · F Pothier · M A Sirard
    Methods in Molecular Biology 02/1995; 48:161-6. DOI:10.1385/0-89603-304-X:161 · 1.29 Impact Factor
  • M GAGNE · F POTHIER · M.-A. Sirard
    Theriogenology 01/1993; 39(1):223-223. DOI:10.1016/0093-691X(93)90078-J · 1.80 Impact Factor
  • M C Pepin · F Pothier · N Barden
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    ABSTRACT: We have created transgenic mouse lines with impaired glucocorticoid receptor function by expression of a type II glucocorticoid receptor antisense RNA in brain tissues. These animals have endocrinological characteristics similar to those seen in depression, including a hyperactive hypothalamic-pituitary-adrenal axis as indicated by elevated plasma corticosterone and adrenocorticotropin hormone levels. Treatment of transgenic animals with the tricyclic antidepressant desipramine increased hypothalamic glucocorticoid receptor mRNA concentration and dexamethasone-binding activity while decreasing plasma adrenocorticotropin hormone concentration and corticosterone levels. These results support the hypothesis that antidepressants exert action on the hypothalamic-pituitary-adrenal axis through modulation of glucocorticoid receptor gene expression.
    Molecular Pharmacology 01/1993; 42(6):991-5. · 4.13 Impact Factor
  • M C Pepin · Francois Pothier · Nicholas Barden
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    ABSTRACT: Glucocorticoids, in conjunction with their cognate receptors, exert negative-feedback effects on the hypothalamus-pituitary-adrenal axis, suppressing adrenal steroid secretions. Two types of corticosteroid receptor, distinguishable by their ability to bind corticosterone, have been identified as classical mineralocorticoid (type I) and glucocorticoid (type II) receptors by cloning their complementary DNAs. The type I receptor controls the basal circadian rhythm of corticosteroid secretion. Both receptor types are involved in negative feedback, but the type II receptor may be more important for terminating the stress response as it is the only one to be increased in animals rendered more sensitive to corticosteroid negative-feedback effects. Here we create a transgenic mouse with impaired corticosteroid-receptor function by partially knocking out gene expression with type II glucocorticoid receptor antisense RNA. We use this animal to study the glucocorticoid feedback effect on the hypothalamus-pituitary-adrenal axis.
    Nature 03/1992; 355(6362):725-8. DOI:10.1038/355725a0 · 41.46 Impact Factor
  • François Pothier · Mariette Ouellet · J P Julien · Sylvain L. Guérin
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    ABSTRACT: We have developed an improved method for determining CAT activity directed by stably (transgenic mice) or transiently (tissue culture cell lines) introduced CAT reporter gene constructs. The procedure is based on the use of a new buffer system which considerably increases the stability of the CAT enzyme during the preparation of the crude cell extracts. When compared to other procedures, our method enables an increase of up to 100-fold in the sensitivity of the assay, depending on the transgenic tissue tested. Furthermore, a strong increase (up to 23-fold) was also observed with various promoter/CAT constructs transiently transfected in established tissue culture cell lines. This increase in sensitivity provides a significant reduction in the time required to perform the CAT assay when strong promoters are studied (from 18 to 1 hr) and is also very useful for the analysis of CAT gene expression driven by weak promoters.
    DNA and Cell Biology 01/1992; 11(1):83-90. DOI:10.1089/dna.1992.11.83 · 2.06 Impact Factor
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    ABSTRACT: Mouse secretory protease inhibitor p12 is significantly transcribed by the cells from the seminal vesicle, the coagulating gland, the ventral prostate, and to a lesser level by the pancreas. It is otherwise undetectable in every other tissue examined. To study the molecular mechanisms involved in this model of cell-specific control of gene expression, we cloned fragments containing various lengths of the p12 promoter upstream of the CAT reporter gene. We demonstrated that p12 sequences from +34 to -108 relative to the CAP site can confer a constitutive level of CAT expression following transient transfection in non-prostatic CV1 and GH4C1 cells. We identified within this minimal p12 promoter the cis-acting sequences needed to direct such a significant level of CAT expression. A DNA-binding site (p12.A) highly homologous to the rat growth hormone (rGH) sequence recognized by the trans-acting factor GC2 was identified between the TATA- and the double CAAT-box sequences from the p12 promoter. Using competition and mutation analysis, we provide evidence that the positively acting p12.A-binding protein is likely to be the rGH GC2 transcription factor, suggesting that the same, or a very similar factor, regulates expression of both rGH and p12 genes. By further analysis of the p12 5'-flanking sequences, we demonstrated that plasmids including sequences from -109 to -843 can strongly repress the level of transcription directed by this minimal p12 promoter, providing evidence for the presence of cis-acting negative regulatory elements critical for the establishment of p12 gene extinction in non-prostatic cells.
    Journal of Biological Chemistry 01/1991; 265(35):22035-43. · 4.57 Impact Factor
  • M Gagné · F Pothier · M.A. Sirard
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    ABSTRACT: This study was designed to investigate the potential use of in vitro matured, in vitro fertilized bovine zygotes for producing transgenic calves by microinjection of foreign DNA. In Experiment 1, the effect of centrifugation (4 min, 15,000 x g, 20 degrees C) on in vitro derived bovine zygotes was evaluated. In vitro development from 2 to 8 cells was not affected (80 vs 78%) when control zygotes (n = 211) were compared with zygotes treated (n = 210) 18 h post insemination. In Experiment 2, the influence of the centrifugation alone on the developmental potential of embryos was evaluated in rabbit oviducts for 120 h. The percentage of control and treated zygotes that developed to 1, 2 to 8, 8 to 32 and more than 32 cells were 7, 54, 10 and 10% vs 7, 40, 11 and 10%, respectively. In Experiment 3, the effect of pronuclear injection with plasmid containing CRF (corticotropin releasing factor) gene or pOCAT 330 Delta 1 plasmids; 2 microg/ml in Tris 10 mM, EDTA 0.2 mM, 18 to 20 h post insemination was evaluated by in vivo development in the rabbit oviduct. The embryos submitted only to centrifugation and vortexing resulted in a morula-blastocyst (> 32 cells) rate of 25% (n = 226) compared with the injected zygotes of which only 5% (n = 206) achieved the same stage. We conclude that in vitro produced bovine zygotes have a reduced developmental potential following microinjection, and this effect is not due to the centrifugation process.
    Theriogenology 08/1990; 34(2):417-25. DOI:10.1016/0093-691X(90)90533-Y · 1.80 Impact Factor

Publication Stats

753 Citations
132.97 Total Impact Points


  • 1993–2012
    • Laval University
      • Department of Animal Sciences
      Quebec City, Quebec, Canada
  • 1990–2001
    • Université du Québec
      • Département des Sciences Animales
      Québec, Quebec, Canada
  • 1995
    • Centre hospitalier de l'Université de Montréal (CHUM)
      Montréal, Quebec, Canada