Abstract
BackgroundThe injection of Neb-colloostatin into T. molitor females causes gonadoinhibitory effects on ovarian development. This peptide inhibits intercellular space formation (patency) in follicular epithelium and results in slowed vitellogenesis, delayed ovulation, reduced number of eggs laid and presumably cell death in the terminal follicles. However, as does the form of cell death in the terminal follicle, the mode of action of Neb-colloostatin remains unknown.ResultsWe tested Neb-colloostatin for a sterilizing effect on females of Tenebrio molitor. We report that injection of nanomolar doses of Neb-colloostatin induce ovarian follicle atresia in 4-day old females during their first gonadotropic cycle. Light microscope observations revealed morphological changes in the ovary: after Neb-colloostatin injection the terminal oocytes are significantly smaller and elicit massive follicle resorption, but the control terminal follicles possess translucent ooplasm in oocytes at different stages of vitellogenesis. A patency is visible in follicular epithelium of the control vitellogenic oocytes, whereas peptide injection inhibits intercellular space formation and, in consequence, inhibits vitellogenesis. Confocal and electron microscope examination showed that peptide injection causes changes in the morphology indicating death of follicular cells. We observed F-actin cytoskeleton disorganization, induction of caspase activity, changes in chromatin organization and autophagic vacuole formation. Moreover, the apical cytoplasm of follicular cells is filled with numerous free ribosomes, probably indicating a higher demand for protein biosynthesis, especially in preparation for autophagic vacuole formation. On the other hand, the process of polyribosomes formation is inhibited, indicating the contributing effect of this hormone.ConclusionNeb-colloostatin induces atresia in the mealworm ovary. Degeneration of T. molitor follicles includes changes in morphology and viability of follicular cells, and oosorption as a consequence of these changes.
Highlights
The injection of Neb-colloostatin into T. molitor females causes gonadoinhibitory effects on ovarian development
Neb-colloostatin injection leads to resorption of vitellogenic ovarian follicles in T. molitor Injection of Neb-colloostatin into the hemocoel of T. molitor females during their first reproductive cycle showed that this peptide strongly inhibited ovarian growth and oocyte development (Figure 1)
Apoptosis and autophagy are involved in follicle cell death after Neb-colloostatin injection After morphological observations, the entire ovaries were stained in order to detect peptide-induced changes in the organization of the F-actin cytoskeleton and chromatin, Figure 1 Light micrographs of ovaries (A-B) and follicles (C-D) of T. molitor
Summary
The injection of Neb-colloostatin into T. molitor females causes gonadoinhibitory effects on ovarian development. Aea-TMOF discovered by Borovsky et al [5] in late vitellogenic ovaries of A. aegypti females as well as Neb-TMOF isolated by Bylemans et al [6,7] from vitellogenic ovaries of the grey fleshfly N. bullata, directly inhibit the biosynthesis of trypsin- and chymotrypsin-like enzymes in the epithelial cells of the insect midgut. This results in the reduction of free amino acids in hemolymph and, in consequence, in the blocking of the biosynthesis of vitellogenin, a protein essential for oocyte growth. Injection of Neb-colloostatin in physiological concentrations results in significant hemocytotoxicity and a marked increase in apoptotic activity in T. molitor haemocytes [14]
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