| 1. |
- Inzunza, José
(författare)
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New micromanipulative techniques in reproductive biology
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- New micro manipulative techniques have facilitated development to help patients who suffer from infertility and spontaneous abortion, as well as those who carry severe genetic diseases and have risks of delivering an affected child. Preimplantation genetic diagnosis (PGD), which is based on obtaining blastomere biopsy from an eight-cell embryo, has made it possible to diagnose severe defects caused by a genetic factor. In the animal laboratory, ovarian transplantation is a new technique that provides a tool in research to save valuable mutants and transgenic animals that have an infertility not caused by germ cells. The infertility can be due to prepubertal lethality, and inability to mate or incapacity to complete a successful fertilization, gestation or parturition. The creation of a technique for transfer of mouse embryos into germ free recipients to produce gnotobiotic mice will facilitate researchers to study the micro flora of transgenic and special mutants more specifically. In this thesis we improved three micro manipulative techniques and the possibilities of applying them in research laboratories and in in-vitro fertilization units. 1) We created an improvement in the embryo biopsy micro technique that is used to allow the preimplantation genetic diagnosis (PGD) using a single-needle to perform the blastomere biopsy. 2) We used the ovarian transplantation micro-surgical technique to save valorous mutants and transgenic animals when the reason for infertility was other than non-functional germ cells. 3) We created a new micro technique to allow transfer of transgenic animals to a germ free state, through the embryo transfer into a germ-free or gnotobiotic recipient. We tested and successfully applied a single-needle approach to obtain blastomere biopsies from human preimplantation embryos for preimplantation genetic diagnosis (PGD). The method was first evaluated in a mouse system and shown to be compatible with a high degree of in-vitro and in-vivo development of biopsied mouse embryos. Biopsied mouse embryos after transfer to recipient mice underwent implantation, normal development and delivery. Successful human preimplantation diagnosis, followed by pregnancies and birth of healthy babies, was established with two out of three couples carrying a risk to transmit chromosomal abnormalities leading to severe diseases. This is the first report of the use of a single-needle approach in human PGD. We evaluated an ovary transplantation method by transplanting ovaries from females belonging to a non-reproductive BALB/cByJ mutant mouse strain. All transplanted mice, BALB/c.C57BL/6By, produced offspring and 94 % of the progeny originated from the transplanted ovaries. The mean litter size and the mating period needed for productive mating to occur were similar to what is observed for corresponding control mice. Subsequently we also performed ovary transplantation of estrogen beta-receptor knockout (ERbeta-/-) ovaries into wild type and normal C57B1/6 recipient mice. The litters' sizes were similar after transplantation of ERbeta-/- and WT ovaries to normal recipients, hence referring to an extra-ovarian cause of subfertility. Lastly we demonstrated that it is possible to obtain germ-free animals using embryo transfer of conventional or transgenic mice to germ-free recipients inside a steel isolator. We have developed a unique embryo transfer technique that will facilitate researchers to use this procedure as a tool in changing the status of different mutants into germ-free mice. All these three techniques are powerful tools in the field of reproductive medicine and biology.
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| 2. |
- Inzunza, Jose, et al.
(författare)
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Selective insulin-like growth factor-I antagonist inhibits mouse embryo development in a dose-dependent manner
- 2010
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Ingår i: Fertility and Sterility. - : Elsevier BV. - 0015-0282 .- 1556-5653. ; 93:8, s. 2621-2626
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To study the role of a synthetic insulin-like growth factor-I receptor (IGF-IR) antagonist, picropodophyllin, for mouse preimplantation embryo development in vivo and in vitro. DESIGN: In vitro and in vivo study. SETTING: Hospital-based research unit. ANIMALS: FVB/N mice and mouse embryos. INTERVENTION(S): The effect of picropodophyllin in mouse embryo development in vivo and in vitro, immunohistochemistry, ELISA, polymerase chain reaction. MAIN OUTCOME MEASURE(S): Embryo development, presence of IGF-IR, messenger RNA expression, IGF-I synthesis. RESULT(S): The effect of picropodophyllin on embryo development in vitro and in vivo was not reversible. Mice treated with picropodophyllin 1 to 3 days after mating had a reduced number of blastocysts, 40.5% versus 78.8%, and a higher number of embryos with delayed development, 48.6% versus 11.5%. Insulin-like growth factor-IR protein is present in both phosphorylated and nonphosphorylated form at all stages of embryo development. The relative IGF-IR messenger RNA expression was highest in the oocyte and reduced during development to blastocyst stage. Insulin-like growth factor-I in culture media was reduced after picropodophyllin treatment. CONCLUSION(S): We conclude that IGF-I has an important role in normal mouse embryo development and that its receptor plays an essential role in the embryonic genome activation process.
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| 3. |
- Lupu, Diana, et al.
(författare)
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Fluoxetine Affects Differentiation of Midbrain Dopaminergic Neurons In Vitro
- 2018
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Ingår i: Molecular Pharmacology. - New York : American Society for Pharmacology and Experimental Therapeutics. - 0026-895X .- 1521-0111. ; 94:4, s. 1220-1231
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Tidskriftsartikel (refereegranskat)abstract
- Recent meta-analyses found an association between prenatal exposure to the antidepressant fluoxetine (FLX) and an increased risk of autism in children. This developmental disorder has been related to dysfunctions in the brains' rewards circuitry, which, in turn, has been linked to dysfunctions in dopaminergic (DA) signaling. The present study investigated if FLX affects processes involved in dopaminergic neuronal differentiation. Mouse neuronal precursors were differentiated into midbrain dopaminergic precursor cells (mDPCs) and concomitantly exposed to clinically relevant doses of FLX. Subsequently, dopaminergic precursors were evaluated for expression of differentiation and stemness markers using quantitative polymerase chain reaction. FLX treatment led to increases in early regional specification markers orthodenticle homeobox 2 (Otx2) and homeobox engrailed-1 and -2 (En1 and En2). On the other hand, two transcription factors essential for midbrain dopaminergic (mDA) neurogenesis, LIM homeobox transcription factor 1 alpha (Lmx1a) and paired-like homeodomain transcription factor 3 (Pitx3) were downregulated by FLX treatment. The stemness marker nestin (Nes) was increased, whereas the neuronal differentiation marker beta 3-tubulin (Tubb3) decreased. Additionally, we observed that FLX modulates the expression of several genes associated with autism spectrum disorder and downregulates the estrogen receptors (ERs) alpha and beta. Further investigations using ER beta knockout (BERKO) mDPCs showed that FLX had no or even opposite effects on several of the genes analyzed. These findings suggest that FLX affects differentiation of the dopaminergic system by increasing production of dopaminergic precursors, yet decreasing their maturation, partly via interference with the estrogen system.
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| 4. |
- Segura-Aguilar, Juan, et al.
(författare)
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Astrocytes protect dopaminergic neurons against aminochrome neurotoxicity
- 2022
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Ingår i: Neural Regeneration Research. - : Medknow. - 1673-5374 .- 1876-7958. ; 17:9, s. 1861-1866
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Forskningsöversikt (refereegranskat)abstract
- Astrocytes protect neurons by modulating neuronal function and survival. Astrocytes support neurons in several ways. They provide energy through the astrocyte-neuron lactate shuttle, protect neurons from excitotoxicity, and internalize neuronal lipid droplets to degrade fatty acids for neuronal metabolic and synaptic support, as well as by their high capacity for glutamate uptake and the conversion of glutamate to glutamine. A recent reported astrocyte system for protection of dopamine neurons against the neurotoxic products of dopamine, such as aminochrome and other o-quinones, were generated under neuromelanin synthesis by oxidizing dopamine catechol structure. Astrocytes secrete glutathione transferase M2-2 through exosomes that transport this enzyme into dopaminergic neurons to protect these neurons against aminochrome neurotoxicity. The role of this new astrocyte protective mechanism in Parkinson´s disease is discussed.
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| 5. |
- Segura-Aguilar, Juan, et al.
(författare)
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Neuroprotection against Aminochrome Neurotoxicity : Glutathione Transferase M2-2 and DT-Diaphorase
- 2022
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Ingår i: Antioxidants. - : MDPI AG. - 2076-3921. ; 11:2
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione is an important antioxidant that plays a crucial role in the cellular protection against oxidative stress and detoxification of electrophilic mutagens, and carcinogens. Glutathione transferases are enzymes catalyzing glutathione-dependent reactions that lead to inactivation and conjugation of toxic compounds, processes followed by subsequent excretion of the detoxified products. Degeneration and loss of neuromelanin-containing dopaminergic neurons in the nigrostriatal neurons generally involves oxidative stress, neuroinflammation, alpha-synuclein aggregation to neurotoxic oligomers, mitochondrial dysfunction, protein degradation dysfunction, and endoplasmic reticulum stress. However, it is still unclear what triggers these neurodegenerative processes. It has been reported that aminochrome may elicit all of these mechanisms and, interestingly, aminochrome is formed inside neuromelanin-containing dopaminergic neurons during neuromelanin synthesis. Aminochrome is a neurotoxic ortho-quinone formed in neuromelanin synthesis. However, it seems paradoxical that the neurotoxin aminochrome is generated during neuromelanin synthesis, even though healthy seniors have these neurons intact when they die. The explanation of this paradox is the existence of protective tools against aminochrome neurotoxicity composed of the enzymes DT-diaphorase, expressed in these neurons, and glutathione transferase M2-2, expressed in astrocytes. Recently, it has been reported that dopaminergic neurons can be protected by glutathione transferase M2-2 from astrocytes, which secrete exosomes containing the protective enzyme.
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| 6. |
- Valdes, Raúl, et al.
(författare)
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Cellular Trafficking of Glutathione Transferase M2-2 Between U373MG and SHSY-S7 Cells is Mediated by Exosomes
- 2021
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Ingår i: Neurotoxicity research. - : Springer Science and Business Media LLC. - 1029-8428 .- 1476-3524. ; 39:2, s. 182-190
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Tidskriftsartikel (refereegranskat)abstract
- The enzyme glutathione transferase M2-2, expressed in human astrocytes, increases its expression in the presence of aminochrome and catalyzes the conjugation of aminochrome, preventing its toxic effects. Secretion of the enzyme glutathione transferase M2-2 from U373MG cells, used as a cellular model for astrocytes, has been reported, and the enzyme is taken up by neuroblastoma SYSH-S7 cells and provide protection against aminochrome. The present study provides evidence that glutathione transferase M2-2 is released in exosomes from U373MG cells, thereby providing a means for intercellular transport of the enzyme. With particular relevance to Parkinson disease and other degenerative conditions, we propose a new mechanism by which astrocytes may protect dopaminergic neurons against the endogenous neurotoxin aminochrome.
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