title: Biochemical changes in the transition from vitellogenesis to follicular atresia in the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae).
abstract: In this work, we have explored the biochemical changes characterizing the transition from vitellogenesis to follicular atresia, employing the hematophagous insect vector Dipetalogaster maxima as a model. Standardized insect rearing conditions were established to induce a gradual follicular degeneration stage by depriving females of blood meal during post-vitellogenesis. For the studies, hemolymph and ovaries were sampled at representative days of pre-vitellogenesis, vitellogenesis and early and late follicular atresia. When examined by scanning electron microscopy, ovarioles at the initial stage of atresia were small but still showed some degree of asynchronism, a feature that was lost in an advanced degeneration state. At late follicular atresia, in vivo uptake assays of fluorescently labeled vitellogenin (Vg-FITC) showed loss of competitiveness of oocytes to uptake vitellogenin. Circulating vitellogenin levels in atresia were significantly higher than those registered at pre-vitellogenesis, most likely to maintain appropriate conditions for another gonotrophic cycle if a second blood meal is available. Follicular atresia was also characterized by partial proteolysis of vitellin, which was evidenced in ovarian homogenates by western blot. When the activity of ovarian peptidases upon hemoglobin (a non-specific substrate) was tested, higher activities were detected at early and late atresia whereas the lowest activity was found at vitellogenesis. The activity upon hemoglobin was significantly inhibited by pepstatin A (an aspartic peptidase inhibitor), and was not affected by E64 (a cysteine peptidase inhibitor) at any tested conditions. The use of specific fluorogenic substrates demonstrated that ovarian homogenates at early follicular atresia displayed high cathepsin D-like activity, whereas no activity of either, cathepsin B or L was detected. Mass spectrometry analysis of the digestion products of the substrate Abz-AIAFFSRQ-EDDnp further confirmed the presence of a cathepsin D-like peptidase in ovarian tissue. In the context of our findings, the early activation of cathepsin D-like peptidase could be relevant in promoting yolk protein recycling and/or enhancing follicle removal.
authors.affiliation: Departamento de Bioquímica Clínica, Centro de Investigaciones en Bioquímica Clínica e Inmunología, Facultad de Ciencias Químicas-Universidad Nacional de Córdoba, Córdoba, CP 5000, Argentina.
authors.forename: Silvina A
authors.initials: SA
authors.lastname: Aguirre
authors.forename: Leonardo L
authors.initials: LL
authors.lastname: Fruttero
authors.forename: Jimena
authors.initials: J
authors.lastname: Leyria
authors.forename: Marina S
authors.initials: MS
authors.lastname: Defferrari
authors.forename: Paulo M
authors.initials: PM
authors.lastname: Pinto
authors.forename: Beatriz P
authors.initials: BP
authors.lastname: Settembrini
authors.forename: Edilberto R
authors.initials: ER
authors.lastname: Rubiolo
authors.forename: Célia R
authors.initials: CR
authors.lastname: Carlini
authors.forename: Lilián E
authors.initials: LE
authors.lastname: Canavoso
mesh_terms: D000818:Animals; D002402:Cathepsin D / Q000378:metabolism; D002853:Chromatography, Liquid; D005260:Female; D005496:Follicular Atresia / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D009865:Oocytes / Q000378:metabolism; D010053:Ovary / Q000201:enzymology / Q000648:ultrastructure; D014225:Triatominae / Q000378:metabolism*; D014818:Vitellogenesis; D014819:Vitellogenins / Q000378:metabolism
chemical_list: D014819:Vitellogenins; D002402:Cathepsin D