The Anthelmintic Quassinoids Ailanthone and Bruceine a Induce Infertility in the Model Organism Caenorhabditis elegans by an Apoptosis-like Mechanism Induced in Gonadal and Spermathecal Tissues

Abstract

In continuation of the search for new anthelmintic natural products, the study at hand investigated the nematicidal effects of the two naturally occurring quassinoids ailanthone and bruceine A against the reproductive system of the model nematode Caenorhabditis elegans to pinpoint their anthelmintic mode of action by the application of various microscopic techniques. Differential Interference Contrast (DIC) and the epifluorescence microscopy experiments used in the presented study indicated the genotoxic effects of the tested quassinoids (c _ailanthone = 50 µM, c _{bruceine A} = 100 µM) against the nuclei of the investigated gonadal and spermathecal tissues, leaving other morphological key features such as enterocytes or body wall muscle cells unimpaired. In order to gain nanoscopic insight into the morphology of the gonads as well as the considerably smaller spermathecae of C. elegans, an innovative protocol of polyethylene glycol embedding, ultra-sectioning, acridine orange staining, tissue identification by epifluorescence, and subsequent AFM-based ultrastructural data acquisition was applied. This sequence allowed the facile and fast assessment of the impact of quassinoid treatment not only on the gonadal but also on the considerably smaller spermathecal tissues of C. elegans. These first-time ultrastructural investigations on C. elegans gonads and spermathecae by AFM led to the identification of specific quassinoid-induced alterations to the nuclei of the reproductive tissues (e.g., highly condensed chromatin, impaired nuclear membrane morphology, as well as altered nucleolus morphology), altogether implying an apoptosis-like effect of ailanthone and bruceine A on the reproductive tissues of C. elegans.

Keywords: Caenorhabditis elegans; ailanthone; anthelmintic natural products; atomic force microscopy; bruceine A; polyethylene glycol embedding; quassinoid; ultramicrotomy; ultrastructural morphology.

Figure 1

Structures of the tested quassinoids ailanthone (1) and bruceine A (2).

Figure 2

Germ line morphology of untreated L4 WT C. elegans hermaphrodites elucidated by Differential Interference Microscopy (DIC). (A): Representative spermathecal region surrounded by natively developing embryos (B): Representative vulvar region surrounded by developing embryos (C): Close up on distal gonadal arm (D): Close up on proximal gonadal arm (E,F): Typical physiological stages of embryo development in an untreated individual. Cut = cuticle, DG = distal gonad, E = egg, Int = intestine, PG = proximal gonad, SP = spermatheca, Vul = vulva.

Figure 2

Germ line morphology of untreated L4 WT C. elegans hermaphrodites elucidated by Differential Interference Microscopy (DIC). (A): Representative spermathecal region surrounded by natively developing embryos (B): Representative vulvar region surrounded by developing embryos (C): Close up on distal gonadal arm (D): Close up on proximal gonadal arm (E,F): Typical physiological stages of embryo development in an untreated individual. Cut = cuticle, DG = distal gonad, E = egg, Int = intestine, PG = proximal gonad, SP = spermatheca, Vul = vulva.

Figure 3

Germ line morphology of bruceine A-treated (100 µM, 48 h) L4 WT C. elegans hermaphrodites elucidated by Differential Interference Microscopy (DIC). (A): Representative DIC close up on vulvar region of a bruceine A-treated L4 hermaphrodite; note the overall missing embryos in the uterus region (B): Representative close up on spermathecal region of a bruceine A-treated C. elegans hermaphrodite; specific morphological impairments were not clearly recognizable by DIC microscopy in single spermathecae. (C): Gonadal arm of bruceine A-treated C. elegans individual; specific morphological impairments were not clearly recognizable by DIC microscopy. Cut = cuticle, DG = distal gonad, Int = intestine, PG = proximal gonad, SP = spermatheca, Vul = vulva.

Figure 3

Germ line morphology of bruceine A-treated (100 µM, 48 h) L4 WT C. elegans hermaphrodites elucidated by Differential Interference Microscopy (DIC). (A): Representative DIC close up on vulvar region of a bruceine A-treated L4 hermaphrodite; note the overall missing embryos in the uterus region (B): Representative close up on spermathecal region of a bruceine A-treated C. elegans hermaphrodite; specific morphological impairments were not clearly recognizable by DIC microscopy in single spermathecae. (C): Gonadal arm of bruceine A-treated C. elegans individual; specific morphological impairments were not clearly recognizable by DIC microscopy. Cut = cuticle, DG = distal gonad, Int = intestine, PG = proximal gonad, SP = spermatheca, Vul = vulva.

Figure 4

High-magnification epifluorescence images of gonadal tissues in acridine orange-stained ultra-sections of quassinoid-treated as well as untreated L4 C. elegans individuals. (A): Representative depiction of untreated gonadal tissue after AO staining; note the prominent yellow-green fluorescence in nuclei of single oocytes (B): Representative depiction of ailanthone-treated gonadal tissues after AO staining; note the reduced fluorescence signal in the nuclei as well as the overall increased red fluorescence in the attendant cytoplasm (C): Representative depiction of brucein A-treated gonadal tissues after AO staining; note the change in overall fluorescence similar to ailanthone-treated gonadal tissues (B). Cut = cuticle, Int = intestine, N = nucleus, O = single oocyte.

Figure 4

High-magnification epifluorescence images of gonadal tissues in acridine orange-stained ultra-sections of quassinoid-treated as well as untreated L4 C. elegans individuals. (A): Representative depiction of untreated gonadal tissue after AO staining; note the prominent yellow-green fluorescence in nuclei of single oocytes (B): Representative depiction of ailanthone-treated gonadal tissues after AO staining; note the reduced fluorescence signal in the nuclei as well as the overall increased red fluorescence in the attendant cytoplasm (C): Representative depiction of brucein A-treated gonadal tissues after AO staining; note the change in overall fluorescence similar to ailanthone-treated gonadal tissues (B). Cut = cuticle, Int = intestine, N = nucleus, O = single oocyte.

Figure 5

High-magnification epifluorescence images of spermathecal tissues in acridine orange-stained ultra-sections of quassinoid-treated as well as untreated L4 C. elegans individuals. (A): Representative depiction of untreated spermathecal tissues after AO staining. (B): Representative depiction of ailanthone-treated spermathecal tissues after AO staining; note the inhomogeneous green fluorescence in single cytoplasmic areas as well as the overall increased granularity of the tissue (C): Representative depiction of brucein A-treated spermathecal tissues after AO staining; note the inhomogeneous green fluorescence in single cytoplasmic areas as well as the overall increased granularity of the tissue. Cut = cuticle, Int = intestine, N = nucleus, SP = single spermatocyte.

Figure 6

AFM-elucidated ultrastructure of C. elegans L4 hermaphrodite´s gonadal tissues after treatment with the quassinoids ailanthone (c _ailanthone = 50 µM) and bruceine A (c _{bruceine A} = 100 µM) and compared to untreated gonadal morphology. (A): Representative native gonadal tissue, single oocytes, and corresponding nuclei are easily detectable (B): Close up on a single untreated oocyte (C): Representative gonadal tissue of ailanthone-treated individual (D): Close up on single ailanthone-treated oocyte; note the specific vacuolization in the gonadal tissue as well as the impaired morphology of nucleus and nucleolus (e.g., highly condensed chromatin (white arrowhead) compared to untreated karyoplasm morphology (A,B)) (E): Representative gonadal tissue of bruceine A-treated individual (F): Close up on single bruceine A-treated oocyte; note the increased vacuolization in the gonadal tissue as well as the impaired morphology of nucleus and nucleolus (e.g., highly condensed chromatin (white arrowhead) compared to untreated karyoplasm morphology (A,B)) (G): Depiction of ailanthone-treated gonadal region in a L4 hermaphrodite, showing the accumulation of mitochondria (white arrowheads) most probably in the rachis region. (H): Close up on gonadal region in a L4 hermaphrodite after ailanthone treatment showing the accumulation of mitochondria (white arrowheads) most probably in the rachis region. Cut = cuticle, N = nucleus, Nuc = nucleolus, O = single oocyte.

Figure 6

AFM-elucidated ultrastructure of C. elegans L4 hermaphrodite´s gonadal tissues after treatment with the quassinoids ailanthone (c _ailanthone = 50 µM) and bruceine A (c _{bruceine A} = 100 µM) and compared to untreated gonadal morphology. (A): Representative native gonadal tissue, single oocytes, and corresponding nuclei are easily detectable (B): Close up on a single untreated oocyte (C): Representative gonadal tissue of ailanthone-treated individual (D): Close up on single ailanthone-treated oocyte; note the specific vacuolization in the gonadal tissue as well as the impaired morphology of nucleus and nucleolus (e.g., highly condensed chromatin (white arrowhead) compared to untreated karyoplasm morphology (A,B)) (E): Representative gonadal tissue of bruceine A-treated individual (F): Close up on single bruceine A-treated oocyte; note the increased vacuolization in the gonadal tissue as well as the impaired morphology of nucleus and nucleolus (e.g., highly condensed chromatin (white arrowhead) compared to untreated karyoplasm morphology (A,B)) (G): Depiction of ailanthone-treated gonadal region in a L4 hermaphrodite, showing the accumulation of mitochondria (white arrowheads) most probably in the rachis region. (H): Close up on gonadal region in a L4 hermaphrodite after ailanthone treatment showing the accumulation of mitochondria (white arrowheads) most probably in the rachis region. Cut = cuticle, N = nucleus, Nuc = nucleolus, O = single oocyte.

Figure 7

AFM-elucidated ultrastructure of C. elegans L4 hermaphrodite´s spermathecal tissues after treatment with the quassinoids ailanthone (c_ailanthone = 50 µM) and bruceine A (c_{bruceine A} = 100 µM) and compared to untreated spermathecal morphology. (A): Representative untreated spermathecal tissue (B): Close up on single spermatocytes (C): Representative ailanthone-treated spermathecal tissue (D): Close up on single spermatid-like cells (E): Representative bruceine A-treated spermathecal tissue (F): Close up on single spermatocytes. Mit = mitochondrium, N = nucleus, Nuc = nucleolus, S = single spermatocyte, Sp = single spermatid.

Figure 7

AFM-elucidated ultrastructure of C. elegans L4 hermaphrodite´s spermathecal tissues after treatment with the quassinoids ailanthone (c_ailanthone = 50 µM) and bruceine A (c_{bruceine A} = 100 µM) and compared to untreated spermathecal morphology. (A): Representative untreated spermathecal tissue (B): Close up on single spermatocytes (C): Representative ailanthone-treated spermathecal tissue (D): Close up on single spermatid-like cells (E): Representative bruceine A-treated spermathecal tissue (F): Close up on single spermatocytes. Mit = mitochondrium, N = nucleus, Nuc = nucleolus, S = single spermatocyte, Sp = single spermatid.