El Modelo Probit de Elección Binomial en Transporte

This work was completed in part, with funding from KSEF-2305-RDE-014 to Edmund Rucker and Gertrude Flora Ribble Graduate Student Fellowship to Thomas Gawriluk. I would like to thank CheMyong Ko (University of Illinois) for the CYP19A-iCre mouse, Kay-Uwe Wagner (University of Nebraska Medical Center) for the CAG-CAT-EGFP mouse, Qingjun Wang (University of Kentucky) for the Atg7-flox mouse, Buck Hales (Southern Illinois University) for the StAR antibody, Wendy Katz (University of Kentucky) for embedding tissues, Vincent Cassone (U. Kentucky) for reagents and use of his lab space, Ashley Seifert (U. Kentucky) for use of his tissue processing system, and Maggie Murphy (U. Kentucky) for help with histology. All of the real-time PCR was completed by Xiaoman Hong and assistance with analysis was done by Lane Christenson (University of Kansas Medical Center).

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Figure 3.1. Cyp19-iCre leads to sufficient recombination in the corpus luteum. (A) Genetic strategy to conditionally knockout Becn1. Numbered boxes correspond to exons with dashed boxes representing engineered insertions and triangles represent LoxP sites. (B) Ovaries from P8.5 CAG-CAT-EGFP females on both WT and Cyp19-iCre backgrounds represented in darkfield and GFP-fluorescence with WT ovaries outlined. (C) Representative immunoblots from granulosa cells 48 hours after PMSG injection. (D) Quantified band intensity relative to Actin for BECN1 and SQSTM1, n=4 for each genotype. Data represents mean with standard error of the mean. (E - H) Representative images showing immunofluoresence for EGFP, indicating Cre recombination, co-stained with DAPI on tissue sections from P8.5 ovaries. Low magnification to see entire ovary (E and F). CL = corpus luteum, AF = antral follicle.

High magnification of corpus luteum to demonstrate recombination in luteal cells. Bars equal 100 μM.

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Figure 3.2. Becn1 is required to maintain pregnancy. (A and B) Immunoblots for P8.5 pregnant corpora lutea (A) and relative quantification (B). * denotes p-value <0.05 for Holm-Sidak post-hoc test versus WT. (B) Day of pregnancy when mothers gave birth for WT (n=22), Becn1 (fl/fl) cKO (n=26) and Atg7 cKO (n=18). Each data point represents one individual and the horizontal line indicates the mean. ** denotes p-value

<0.001 for Dunn’s multiple comparison test versus WT. (C and D) Number of implanted fetuses observed (C) and wet mass of both ovaries on P8.5 for individual strains (D).

Data represent mean with s.e.m. (E) Histogram representing the day of parturition observed for WT (n=22) and Becn1(fl/fl) cKO (n=26) females. Each data point represents a single pregnancy, bar is mean, * denotes p-value <0.01 for t-test.

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Figure 3.2 continued. (F) Percent of Becn1(fl/Δ) cKO females that are pregnant on the designated day after mating. Observed number of pregnant females out of total females mated indicated vertically inside each bar. * denotes p-value <0.05 for fisher’s exact test between the two genotypes.

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Figure 3.3. Becn1 cKO early parturition is progesterone-dependent.

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Figure 3.3. Continued. (A) Progesterone quantified from plasma every other day throughout pregnancy for WT, Becn1(fl/fl) cKO and Becn1(fl/Δ) cKO. Data represents mean with s.e.m. * denote a p-value <0.05 for Holm-Sidak post-hoc test versus WT for that day. (B) Diagram depicting dosing paradigm for exogenous progesterone

administration. (C) Day of parturition for females that were given sesame oil (vehicle) or 1 mg progesterone daily (n ≥ 3 for each group). Data represents mean with standard error of the mean. * denotes p-value < 0.001 for Holm-Sidak post-hoc test for interaction indicated. ns = not significant

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Figure 3.4. Becn1 (fl/fl) cKO hormone profile and estrus cyclicity are normal. (A) Percent of time that each estrus stage was observed for WT (n=20) and Becn1 (fl/fl) cKO (n=15) over 21 days. Data represents mean with standard error of the mean. p-value equals 0.09 for two-way ANOVA for genotype x stage interaction. (B, C, D and E) Reproductive hormones progesterone (B), estradiol (C), follicle-stimulating hormone (D) and luteinizing hormone (E) quantified from serum collected during euthanasia at the specified times. For all charts, data represents mean with standard error of mean. * denotes a p-value <0.05, respectively for Holm-Sidak post-hoc test versus WT. ns = not significant.

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Figure 3.5. First eight days of pregnancy plasma progesterone in Becn1 (fl/Δ) cKO. Plasma concentrations of progesterone for Becn1 (fl/Δ) cKO (n=4) and WT (n=6) from P1.5 to P8.5. Data represent mean with standard error of the mean. No significant differences identified, two-tailed t-test for each day, p>0.05.

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Figure 3.6. Becn1 promotes neutral lipid stores in luteal cells. (A – H) Representative images of frozen tissue sections stained with oil red-O (red) and counterstained with hematoxylin (blue) at P8.5 (A-D) and P13.5 (E-H).

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Figure 3.6. Continued. Low magnification indicates staining in medulla, theca and corpus lutea (A, B, E, F). Higher magnification of corpus luteum to highlight luteal cell staining (C, D, E, H). Oil red-O stains neutral lipids, mainly observed as lipid droplets (arrows). Bars equal 100 μM.

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Figure 3.7. Becn1 is necessary for the clearance of GFP-positive vesicles Representative images of frozen tissue sections directly viewed for GFP-LC3. Low magnification (A, B, E and F) shows that only CLs of Becn1 (fl/Δ) cKO have intense GFP. GFP-LC3 puncta are present in Becn1 (fl/Δ) cKO (D and H) but not in WT luteal cells (C and G). Bars equal 100 μM.

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Figure 3.8. Becn1 is necessary for the clearance of autophagosomes and endosomes.

Representative images from transmission electron microscopy of P13.5 large luteal cells from WT (A, C, E) and Becn1 (fl/Δ) cKO (B, D, F). An entire cells ultrastructure where lipid droplets mitochondria and nuclei are seen (A and B). High magnification images indicating mitochondria (Mt), lipid droplets (LD), multi-vesicular bodies (MVB), endosome (End), and autophagosomes (Aut) (C, D, E, F). Scale indicated on lower right corner of each image.

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Figure 3.9. Atg7 ablation have normal gestation and have increased progesterone throughout pregnancy. (A) Genetic strategy to conditionally knockout Atg7. Numbered boxes correspond to exons with dashed boxes representing engineered insertions, ** is a translational STOP and triangles represent LoxP sites. (B and C) Immunofluoresence for EGFP, indicating Cre-recombination in P8.5 ovaries (B) and CL (C). Bar equals 100 μM. (D) Relative quantification of indicated proteins from immunoblots. * denotes p<0.05 for t-test, data represents mean ± s.e.m. (E) Histogram depicting the day of parturition for WT (n=22) and Atg7 cKO (n=18) females. (F) Plasma concentrations for progesterone throughout pregnancy. * denotes p<0.05 for t-test, data represent mean ± s.e.m.

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Figure 3.10 Becn1 cKO and Atg7 cKO have different responses in the progesterone gene network.

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Figure 3.10. Continued. (A, B and C) mRNA expression relative to WT and normalized to Beta-actin and Gapdh for genes for progesterone synthesis (A), cholesterol transport and synthesis (B) and luteal cell signaling (C). Data represents mean with standard error of the mean for n≥4 for each group. * and ** denotes a p-value

<0.05 and <0.001, respectively for Holm-Sidak post-hoc test versus WT individually for each gene.

Copyright © Thomas Raymond Gawriluk 2014

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CHAPTER 4: AUTOPHAGY IS NECESSARY FOR ADULT SERTOLI CELL