Whole-organ isolation approach as a basis for tissue-specific analyses in Schistosoma mansoni

Abstract

Background: Schistosomiasis is one of the most important parasitic diseases worldwide, second only to malaria. Schistosomes exhibit an exceptional reproductive biology since the sexual maturation of the female, which includes the differentiation of the reproductive organs, is controlled by pairing. Pathogenicity originates from eggs, which cause severe inflammation in their hosts. Elucidation of processes contributing to female maturation is not only of interest to basic science but also considering novel concepts combating schistosomiasis.

Methodology/principal findings: To get direct access to the reproductive organs, we established a novel protocol using a combined detergent/protease-treatment removing the tegument and the musculature of adult Schistosoma mansoni. All steps were monitored by scanning electron microscopy (SEM) and bright-field microscopy (BF). We focused on the gonads of adult schistosomes and demonstrated that isolated and purified testes and ovaries can be used for morphological and structural studies as well as sources for RNA and protein of sufficient amounts for subsequent analyses such as RT-PCR and immunoblotting. To this end, first exemplary evidence was obtained for tissue-specific transcription within the gonads (axonemal dynein intermediate chain gene SmAxDynIC; aquaporin gene SmAQP) as well as for post-transcriptional regulation (SmAQP).

Conclusions/significance: The presented method provides a new way of getting access to tissue-specific material of S. mansoni. With regard to many still unanswered questions of schistosome biology, such as elucidating the molecular processes involved in schistosome reproduction, this protocol provides opportunities for, e.g., sub-transcriptomics and sub-proteomics at the organ level. This will promote the characterisation of gene-expression profiles, or more specifically to complete knowledge of signalling pathways contributing to differentiation processes, so discovering involved molecules that may represent potential targets for novel intervention strategies. Furthermore, gonads and other tissues are a basis for cell isolation, opening new perspectives for establishing cell lines, one of the tools desperately needed in the post-genomic era.

Figure 1. Schematic illustration and surface electron microscopy (SEM)-analyses of tegument solubilisation by TS-solution treatment.

A) Untreated control males (upper left) and females (upper right) showing intact tegument (TE) with spines (SP), pits (PI), and sensory endings (SE). B) The tegument was completely removed due to detergent treatment exposing the outer circular muscles (CM) and the basis of the (male-specific) tubercles (TU). Membranocalyx (MC), plasma membrane (PM), longitudinal muscles (LM), basal membrane (BM), musculature (MU), parenchyma (PA); modified according to ; dashed arrow = continued in Figure 2.

Figure 2. Schematic illustration and bright-field microscopy (BF) of gonad tissues following tegument solubilisation and protease treatment.

A) Crude preparation of intact testes (TE) together with a part of an incompletely digested male worm body (MB) and different types of cells (CE) (left) and an mature ovary (Om) surrounded mainly by S4-vitelline cells (VC) from the vitellarium (right); immature ovary (Oi) and ootype (OT) with vitelloduct (VD) and oviduct (OD) isolated from a unisexual female; the ootype was contrasted by brief staining with Ponceau S; asterisk: “hymen”-like morphological structure typical for ootypes of unisexual females , B) Mechanical transfer by pipetting led to the enrichment of pure testes (TE), mature ovaries (Om) after collecting and concentrating. TL (testes lobe), Op (ovary - posterior part containing mature primary oocytes in the case of mature ovaries), Oa (ovary - anterior part containing immature, stem cell-like oogonia); vitellarium (VI) with vitelline lobes (VL); dashed arrow = continued from Figure 1.

Figure 3. Vital staining of isolated reproductive organs.

As an example one testis (left) and one ovary (right) derived from bisexual adult worms are shown, which were stained with 0.4% Trypan Blue immediately after isolation and examined by bright-field microscopy.

Figure 4. Quantitative and qualitative microfluid analysis of total RNA.

RNA-analyses exemplarily shown for RNA isolated from adult males (A), testes (B), and ovaries (C) obtained by the organ isolation procedure were used. The figure shows a “gel-like image” consisting of the RNA-ladder and the appropriate total RNA sample (left) and the corresponding electropherogram (right); fluorescent units (FU), retention time (s).

Figure 5. Gonad-RNA specific RT-PCRs.

Total RNA of testes (Te), ovaries (Ov) and adult couples (merged Mars/Venus symbol) was isolated by Trizol and reverse transcribed. RT-PCRs were performed using gene-specific primers targeting SmHSP70 (Heat shock protein 70), SmFKBP12 (FK506-binding protein), SmCNA (Calcineurin subunit A), SmTGFβRI (Transforming growth factor β receptor I), SmAxDynIC (Axonemal dynein intermediate chain), SmAQP (Aquaporin), SmSPRM1hc (Permease 1 heavy chain), and SmNPP-5 (Nucleotide pyrophosphatase/phosphosdiesterase type 5); for references see Table 1. Marker (M) = Hyperladder I (Bioline). Target genes depicted in green were also analysed by immunoblotting (Figure 7).

Figure 6. Protein patterns of S. mansoni organs/tissues and adults.

1.2 µg total protein from male (Mars symbol), female (Venus symbol), testes (Te), ovaries (Ov), and male tegument (T) were separated by 13% SDS-PAGE and visualised by silver staining. Marker (M) = PageRuler Plus Prestained Protein Ladder (Fermentas).

Figure 7. Gonad protein-specific immunoblots.

15 µg of total protein per lane isolated from adult worms (Mars and Venus symbol), testes (Te), ovaries (Ov), and tegumental proteins of both genders (T) were analysed by immunoblotting employing immune sera directed against SmSPRM1hc (Permease 1 heavy chain), SmHSP70 (Heat shock protein 70), SmAQP (Aquaporin), and SmFKBP12 (FK506-binding protein); for references see Table 1.